Genetic products differentially expressed in tumors and the use thereof

ABSTRACT

The present technology relates to the identification of genetic products expressed in association with tumors and to coding nucleic acids for the expressed products. An embodiment of the present technology also relates to the therapy and diagnosis of disease in which the genetic products are aberrantly expressed in association with tumors, proteins, polypeptides and peptides which are expressed in association with tumors, and to the nucleic acids coding for the polypeptides, peptides and proteins.

This application is a divisional application of U.S. patent applicationSer. No. 14/821,411, which was filed Aug. 7, 2015 (now abandoned), whichis a divisional application of U.S. patent application Ser. No.14/043,109, which was filed Oct. 1, 2013 (now abandoned), which is adivisional application of U.S. patent application Ser. No. 12/423,153,filed Apr. 14, 2009 (now U.S. Pat. No. 8,586,047), which is acontinuation application of U.S. patent application Ser. No. 12/326,997(now U.S. Pat. No. 8,088,588), which was filed on Dec. 3, 2008 and is acontinuation application of U.S. patent application Ser. No. 10/537,002(now U.S. Pat. No. 7,527,933), which was filed on May 20, 2005, which isa National Stage Entry of PCT/EP03/13091, which was filed on Nov. 21,2003 and claimed priority of German Patent Application Number 102 5401.0, which was filed on Nov. 22, 2002. The contents of U.S. patentapplication Ser. Nos. 10/537,002 and 12/326,997, international patentapplication number PCT/EP03/13092, and German Patent Application Number102 54 601.0 are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Despite interdisciplinary approaches and exhaustive use of classicaltherapeutic procedures, cancers are still among the leading causes ofdeath. More recent therapeutic concepts aim at incorporating thepatient's immune system into the overall therapeutic concept by usingrecombinant tumor vaccines and other specific measures such as antibodytherapy. A prerequisite for the success of such a strategy is therecognition of tumor-specific or tumor-associated antigens or epitopesby the patient's immune system whose effector functions are to beinterventionally enhanced. Tumor cells biologically differ substantiallyfrom their nonmalignant cells of origin. These differences are due togenetic alterations acquired during tumor development and result, interalia, also in the formation of qualitatively or quantitatively alteredmolecular structures in the cancer cells. Tumor-associated structures ofthis kind which are recognized by the specific immune system of thetumor-harboring host are referred to as tumor-associated antigens. Thespecific recognition of tumor-associated antigens involves cellular andhumoral mechanisms which are two functionally interconnected units: CD4⁺and CD8⁺ T lymphocytes recognize the processed antigens presented on themolecules of the MHC (major histocompatibility complex) classes II andI, respectively, while B lymphocytes produce circulating antibodymolecules which bind directly to unprocessed antigens. The potentialclinical-therapeutical importance of tumor-associated antigens resultsfrom the fact that the recognition of antigens on neoplastic cells bythe immune system leads to the initiation of cytotoxic effectormechanisms and, in the presence of T helper cells, can cause eliminationof the cancer cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, acentral aim of tumor immunology is to molecularly define thesestructures. The molecular nature of these antigens has been enigmaticfor a long time. Only after development of appropriate cloningtechniques has it been possible to screen cDNA expression libraries oftumors systematically for tumor-associated antigens by analyzing thetarget structures of cytotoxic T lymphocytes (CTL) (van der Bruggen etal., Science 254:1643-7, 1991) or by using circulating autoantibodies(Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes. To thisend, cDNA expression libraries were prepared from fresh tumor tissue andrecombinantly expressed as proteins in suitable systems. Immunoeffectorsisolated from patients, namely CTL clones with tumor-specific lysispatterns, or circulating autoantibodies were utilized for cloning therespective antigens.

In recent years a multiplicity of antigens have been defined in variousneoplasias by these approaches. However, the probes utilized for antigenidentification in the classical methods illustrated above areimmunoeffectors (circulating autoantibodies or CTL clones) from patientsusually having already advanced cancer. A number of data indicate thattumors can lead, for example, to tolerization and anergization of Tcells and that, during the course of the disease, especially thosespecificities which could cause effective immune recognition are lostfrom the immunoeffector repertoire. Current patient studies have not yetproduced any solid evidence of a real action of the previously found andutilized tumor-associated antigens. Accordingly, it cannot be ruled outthat proteins evoking spontaneous immune responses are the wrong targetstructures.

BRIEF SUMMARY OF THE INVENTION

It was the object of the present technology to provide target structuresfor a diagnosis and therapy cancers.

According to the present technology, this object is achieved by thesubject matter of the claims.

According to the present technology, a strategy for identifying andproviding antigens expressed in association with a tumor and the nucleicacids coding therefor was pursued. This strategy is based on the factthat particular genes which are expressed in an organ specific manner,e.g. exclusively in colon, lung or kidney tissue, are reactivated alsoin tumor cells of the respective organs and moreover in tumor cells ofother tissues in an ectopic and forbidden manner. First, data miningproduces a list as complete as possible of all known organ-specificgenes which are then evaluated for their aberrant activation indifferent tumors by expression analyses by means of specific RT-PCR.Data mining is a known method of identifying tumor-associated genes. Inthe conventional strategies, however, transcriptoms of normal tissuelibraries are usually subtracted electronically from tumor tissuelibraries, with the assumption that the remaining genes aretumor-specific (Schmitt et al., Nucleic Acids Res. 27:4251-60, 1999;Vasmatzis et al., Proc. Natl. Acad. Sci. USA. 95:300-4, 1998; Scheurleet al., Cancer Res. 60:4037-43, 2000).

The concept of the present technology, which has proved much moresuccessful, however, is based on utilizing data mining forelectronically extracting all organ-specific genes and then evaluatingsaid genes for expression in tumors.

The present technology thus relates in one aspect to a strategy foridentifying tissue-specific genes differentially expressed in tumors.Said strategy combines data mining of public sequence libraries (“insilico”) with subsequent evaluating laboratory-experimental (“wetbench”) studies.

According to the present technology, a combined strategy based on twodifferent bioinformatic scripts enabled new tumor genes to beidentified. These have previously been classified as being purelyorgan-specific. The finding that these genes are aberrantly activated intumor cells allows them to be assigned a substantially new quality withfunctional implications. According to the present technology, thesetumor-associated genes and the genetic products encoded thereby wereidentified and provided independently of an immunogenic action.

The tumor-associated antigens identified according to the presenttechnology have an amino acid sequence encoded by a nucleic acid whichis selected from the group consisting of (a) a nucleic acid whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1-8, 41-44, 51-59, 84, 117, and 119, a part or derivativethereof, (b) a nucleic acid which hybridizes with the nucleic acid of(a) under stringent conditions, (c) a nucleic acid which is degeneratewith respect to the nucleic acid of (a) or (b), and (d) a nucleic acidwhich is complementary to the nucleic acid of (a), (b) or (c). In apreferred embodiment, a tumor-associated antigen identified according tothe present technology has an amino acid sequence encoded by a nucleicacid which is selected from the group consisting of SEQ ID NOs: 1-8,41-44, 51-59, 84, 117, and 119. In a further preferred embodiment, atumor-associated antigen identified according to the present technologycomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 9-19, 45-48, 60-66, 85, 90-97, 100-102, 105, 106, 111-116,118, 120, 123, 124, and 135-137, a part or derivative thereof.

The present technology generally relates to the use of tumor-associatedantigens identified according to the present technology or of parts orderivatives thereof, of nucleic acids coding therefor or of nucleicacids directed against said coding nucleic acids or of antibodiesdirected against the tumor-associated antigens identified according tothe present technology or parts or derivatives thereof for therapy anddiagnosis. This utilization may relate to individual but also tocombinations of two or more of these antigens, functional fragments,nucleic acids, antibodies, etc., in one embodiment also in combinationwith other tumor-associated genes and antigens for diagnosis, therapyand progress control.

Preferred diseases for a therapy and/or diagnosis are those in which oneor more of the tumor-associated antigens identified according to thepresent technology are selectively expressed or abnormally expressed.

The present technology also relates to nucleic acids and geneticproducts which are expressed in association with a tumor cell.

Furthermore, the present technology relates to genetic products, i.e.nucleic acids and proteins or peptides, which are produced by alteredsplicing (splice variants) of known genes or altered translation usingalternative open reading frames. In this aspect the present technologyrelates to nucleic acids which comprise a nucleic acid sequence selectedfrom the group consisting of sequences according to SEQ ID NOs: 3-5 ofthe sequence listing. Moreover, in this aspect, the present technologyrelates to proteins or peptides which comprise an amino acid sequenceselected from the group consisting of the sequences according to SEQ IDNOs: 10 and 12-14 of the sequence listing. The splice variants of thepresent technology can be used according to the present technology astargets for diagnosis and therapy of tumor diseases.

In particular, the present technology relates to the amino acid sequenceaccording to SEQ ID NO: 10 of the sequence listing which is encoded byan alternative open reading frame identified according to the presenttechnology and differs from the previously described protein sequence(SEQ ID NO: 9) in additional 85 amino acids at the N terminus of theprotein.

Very different mechanisms may cause splice variants to be produced, forexample

-   -   utilization of variable transcription initiation sites    -   utilization of additional exons    -   complete or incomplete splicing out of single or two or more        exons,    -   splice regulator sequences altered via mutation (deletion or        generation of new donor/acceptor sequences),    -   incomplete elimination of intron sequences.

Altered splicing of a gene results in an altered transcript sequence(splice variant). Translation of a splice variant in the region of itsaltered sequence results in an altered protein which may be distinctlydifferent in the structure and function from the original protein.Tumor-associated splice variants may produce tumor-associatedtranscripts and tumor-associated proteins/antigens. These may beutilized as molecular markers both for detecting tumor cells and fortherapeutic targeting of tumors. Detection of tumor cells, for examplein blood, serum, bone marrow, sputum, bronchial lavage, bodilysecretions and tissue biopsies, may be carried out according to thepresent technology, for example, after extraction of nucleic acids byPCR amplification with splice variant-specific oligonucleotides. Inparticular, pairs of primers are suitable as oligonucleotides at leastone of which binds to the region of the splice variant which istumor-associated under stringent conditions. According to the presenttechnology, oligonucleotides described for this purpose in the examplesare suitable, in particular oligonucleotides which have or comprise asequence selected from SEQ ID NOs: 34-36, 39, 40, and 107-110 of thesequence listing. According to the present technology, allsequence-dependent detection systems are suitable for detection. Theseare, apart from PCR, for example gene chip/microarray systems, Northernblot, RNAse protection assays (RDA) and others. All detection systemshave in common that detection is based on a specific hybridization withat least one splice variant-specific nucleic acid sequence. However,tumor cells may also be detected according to the present technology byantibodies which recognize a specific epitope encoded by the splicevariant. Said antibodies may be prepared by using for immunizationpeptides which are specific for said splice variant. In this aspect, thepresent technology relates, in particular, to peptides which have orcomprise a sequence selected from SEQ ID NOs: 17-19, 111-115, 120, and137 of the sequence listing and specific antibodies which are directedthereto. Suitable for immunization are particularly the amino acidswhose epitopes are distinctly different from the variant(s) of thegenetic product, which is (are) preferably produced in healthy cells.Detection of the tumor cells with antibodies may be carried out here ona sample isolated from the patient or as imaging with intravenouslyadministered antibodies. In addition to diagnostic usability, splicevariants having new or altered epitopes are attractive targets forimmunotherapy. The epitopes of the present technology may be utilizedfor targeting therapeutically active monoclonal antibodies or Tlymphocytes. In passive immunotherapy, antibodies or T lymphocytes whichrecognize splice variant-specific epitopes are adoptively transferredhere. As in the case of other antigens, antibodies may be generated alsoby using standard technologies (immunization of animals, panningstrategies for isolation of recombinant antibodies) with utilization ofpolypeptides which include these epitopes. Alternatively, it is possibleto utilize for immunization nucleic acids coding for oligo- orpolypeptides which contain said epitopes. Various techniques for invitro or in vivo generation of epitope-specific T lymphocytes are knownand have been described in detail (for example Kessler J H, et al. 2001,Sahin et al., 1997) and are likewise based on utilizing oligo- orpolypeptides which contain the splice variant-specific epitopes ornucleic acids coding for said oligo- or polypeptides. Oligo- orpolypeptides which contain the splice variant-specific epitopes ornucleic acids coding for said polypeptides may also be used aspharmaceutically active substances in active immunotherapy (vaccination,vaccine therapy).

The present technology also describes proteins which differ in natureand degree of their secondary modifications in normal and tumor tissue(for example Durand & Seta, 2000; Clin. Chem. 46: 795-805; Hakomori,1996; Cancer Res. 56: 5309-18).

The analysis of protein modifications can be done in Western blots. Inparticular, glycosylations which as a rule have a size of several kDaresult in a higher overall mass of the target protein which can beseparated in an SDS-PAGE. For the detection of specific 0- andN-glycosidic bonds protein lysates are incubated with O- orN-glycosylases (according to the instructions of the respectivemanufactures, for example, PNgase, endoglycosidase F, endoglycosidase H,Roche Diagnostics) prior to denaturation using SDS. Thereafter, aWestern blot is performed. If the size of target protein is reduced aspecific glycosylation can be detected in this manner followingincubation with a glycosidase and thus, also the tumor specificity of amodification can be analyzed. Protein regions which are differentiallyglycosylated in tumor cells and healthy cells are of particularinterest. Such differences in glycosylation, however, have hitherto onlybeen described for a few cell surface proteins (for example, Muc1).

According to the present technology, it was possible to detect adifferential glycosylation for Claudin-18 in tumors. Gastrointestinalcarcinomas, pancreas carcinomas, esophagus tumors, prostate tumors aswell as lung tumors have a form of Claudin-18 which is glycosylated at alower level. Glycosylation in healthy tissues masks protein epitopes ofClaudin-18 which are not covered on tumor cells due to lackingglycosylation. Correspondingly it is possible according to the presenttechnology to select ligands and antibodies which bind to these domains.Such ligands and antibodies according to the present technology do notbind to Claudin-18 on healthy cells since here the epitopes are covereddue to glycosylation.

As has been described above for protein epitopes which are derived fromtumor-associated splice variants it is thus possible to use thedifferential glycosylation to distinguish normal cells and tumor cellswith diagnostic as well as therapeutic intention.

In one aspect, the present technology relates to a pharmaceuticalcomposition comprising an agent which recognizes the tumor-associatedantigen identified according to the present technology and which ispreferably selective for cells which have expression or abnormalexpression of a tumor-associated antigen identified according to thepresent technology. In particular embodiments, said agent may causeinduction of cell death, reduction in cell growth, damage to the cellmembrane or secretion of cytokines and preferably have atumor-inhibiting activity. In one embodiment, the agent is an antisensenucleic acid which hybridizes selectively with the nucleic acid codingfor the tumor-associated antigen. In a further embodiment, the agent isan antibody which binds selectively to the tumor-associated antigen, inparticular a complement-activated or toxin conjugated antibody whichbinds selectively to the tumor-associated antigen. In a furtherembodiment, the agent comprises two or more agents which eachselectively recognize different tumor-associated antigens, at least oneof which is a tumor-associated antigen identified according to thepresent technology. Recognition needs not be accompanied directly withinhibition of activity or expression of the antigen. In this aspect ofthe present technology, the antigen selectively limited to tumorspreferably serves as a label for recruiting effector mechanisms to thisspecific location. In a preferred embodiment, the agent is a cytotoxic Tlymphocyte which recognizes the antigen on an HLA molecule and lyses thecells labeled in this way. In a further embodiment, the agent is anantibody which binds selectively to the tumor-associated antigen andthus recruits natural or artificial effector mechanisms to said cell. Ina further embodiment, the agent is a T helper lymphocyte which enhanceseffector functions of other cells specifically recognizing said antigen.

In one aspect, the present technology relates to a pharmaceuticalcomposition comprising an agent which inhibits expression or activity ofa tumor-associated antigen identified according to the presenttechnology. In a preferred embodiment, the agent is an antisense nucleicacid which hybridizes selectively with the nucleic acid coding for thetumor-associated antigen. In a further embodiment, the agent is anantibody which binds selectively to the tumor-associated antigen. In afurther embodiment, the agent comprises two or more agents which eachselectively inhibit expression or activity of different tumor-associatedantigens, at least one of which is a tumor-associated antigen identifiedaccording to the present technology.

The present technology furthermore relates to a pharmaceuticalcomposition which comprises an agent which, when administered,selectively increases the amount of complexes between an HLA moleculeand a peptide epitope from the tumor-associated antigen identifiedaccording to the present technology. In one embodiment, the agentcomprises one or more components selected from the group consisting of(i) the tumor-associated antigen or a part thereof, (ii) a nucleic acidwhich codes for said tumor-associated antigen or a part thereof, (iii) ahost cell which expresses said tumor-associated antigen or a partthereof, and (iv) isolated complexes between peptide epitopes from saidtumor-associated antigen and an MHC molecule. In one embodiment, theagent comprises two or more agents which each selectively increase theamount of complexes between MHC molecules and peptide epitopes ofdifferent tumor-associated antigens, at least one of which is atumor-associated antigen identified according to the present technology.

The present technology furthermore relates to a pharmaceuticalcomposition which comprises one or more components selected from thegroup consisting of (i) a tumor-associated antigen identified accordingto the present technology or a part thereof, (ii) a nucleic acid whichcodes for a tumor-associated antigen identified according to the presenttechnology or for a part thereof, (iii) an antibody which binds to atumor-associated antigen identified according to the present technologyor to a part thereof, (iv) an antisense nucleic acid which hybridizesspecifically with a nucleic acid coding for a tumor-associated antigenidentified according to the present technology, (v) a host cell whichexpresses a tumor-associated antigen identified according to the presenttechnology or a part thereof, and (vi) isolated complexes between atumor-associated antigen identified according to the present technologyor a part thereof and an HLA molecule.

A nucleic acid coding for a tumor-associated antigen identifiedaccording to the present technology or for a part thereof may be presentin the pharmaceutical composition in an expression vector andfunctionally linked to a promoter.

A host cell present in a pharmaceutical composition of the presenttechnology may secrete the tumor-associated antigen or the part thereof,express it on the surface or may additionally express an HLA moleculewhich binds to said tumor-associated antigen or said part thereof. Inone embodiment, the host cell expresses the HLA molecule endogenously.In a further embodiment, the host cell expresses the HLA molecule and/orthe tumor-associated antigen or the part thereof in a recombinantmanner. The host cell is preferably nonproliferative. In a preferredembodiment, the host cell is an antigen-presenting cell, in particular adendritic cell, a monocyte or a macrophage.

An antibody present in a pharmaceutical composition of the presenttechnology may be a monoclonal antibody. In further embodiments, theantibody is a chimeric or humanized antibody, a fragment of a naturalantibody or a synthetic antibody, all of which may be produced bycombinatory techniques. The antibody may be coupled to a therapeuticallyor diagnostically useful agent.

An antisense nucleic acid present in a pharmaceutical composition of thepresent technology may comprise a sequence of 6-50, in particular 10-30,15-30 and 20-30, contiguous nucleotides of the nucleic acid coding forthe tumor-associated antigen identified according to the presenttechnology.

In further embodiments, a tumor-associated antigen, provided by apharmaceutical composition of the present technology either directly orvia expression of a nucleic acid, or a part thereof binds to MHCmolecules on the surface of cells, said binding preferably causing acytolytic response and/or inducing cytokine release.

A pharmaceutical composition of the present technology may comprise apharmaceutically compatible carrier and/or an adjuvant. The adjuvant maybe selected from saponin, GM-CSF, CpG nucleotides, RNA, a cytokine or achemokine. A pharmaceutical composition of the present technology ispreferably used for the treatment of a disease characterized byselective expression or abnormal expression of a tumor-associatedantigen. In a preferred embodiment, the disease is cancer.

The present technology furthermore relates to methods of treating ordiagnosing a disease characterized by expression or abnormal expressionof one of more tumor-associated antigens. In one embodiment, thetreatment comprises administering a pharmaceutical composition of thepresent technology.

In one aspect, the present technology relates to a method of diagnosinga disease characterized by expression or abnormal expression of atumor-associated antigen identified according to the present technology.The method comprises detection of (i) a nucleic acid which codes for thetumor-associated antigen or of a part thereof and/or (ii) detection ofthe tumor-associated antigen or of a part thereof, and/or (iii)detection of an antibody to the tumor-associated antigen or to a partthereof and/or (iv) detection of cytotoxic or T helper lymphocytes whichare specific for the tumor-associated antigen or for a part thereof in abiological sample isolated from a patient. In particular embodiments,detection comprises (i) contacting the biological sample with an agentwhich binds specifically to the nucleic acid coding for thetumor-associated antigen or to the part thereof, to saidtumor-associated antigen or said part thereof, to the antibody or tocytotoxic or T helper lymphocytes specific for the tumor-associatedantigen or parts thereof, and (ii) detecting the formation of a complexbetween the agent and the nucleic acid or the part thereof, thetumor-associated antigen or the part thereof, the antibody or thecytotoxic or T helper lymphocytes. In one embodiment, the disease ischaracterized by expression or abnormal expression of two or moredifferent tumor-associated antigens and detection comprises detection oftwo or more nucleic acids coding for said two or more differenttumor-associated antigens or of parts thereof, detection of two or moredifferent tumor-associated antigens or of parts thereof, detection oftwo or more antibodies binding to said two or more differenttumor-associated antigens or to parts thereof or detection of two ormore cytotoxic or T helper lymphocytes specific for said two or moredifferent tumor-associated antigens. In a further embodiment, thebiological sample isolated from the patient is compared to a comparablenormal biological sample.

In a further aspect, the present technology relates to a method fordetermining regression, course or onset of a disease characterized byexpression or abnormal expression of a tumor-associated antigenidentified according to the present technology, which method comprisesmonitoring a sample from a patient who has said disease or is suspectedof falling ill with said disease, with respect to one or more parametersselected from the group consisting of (i) the amount of nucleic acidwhich codes for the tumor-associated antigen or of a part thereof, (ii)the amount of the tumor-associated antigen or a part thereof, (iii) theamount of antibodies which bind to the tumor-associated antigen or to apart thereof, and (iv) the amount of cytolytic T cells or T helper cellswhich are specific for a complex between the tumor-associated antigen ora part thereof and an MHC molecule. The method preferably comprisesdetermining the parameter(s) in a first sample at a first point in timeand in a further sample at a second point in time and in which thecourse of the disease is determined by comparing the two samples. Inparticular embodiments, the disease is characterized by expression orabnormal expression of two or more different tumor-associated antigensand monitoring comprises monitoring (i) the amount of two or morenucleic acids which code for said two or more different tumor-associatedantigens or of parts thereof, and/or (ii) the amount of said two or moredifferent tumor-associated antigens or of parts thereof, and/or (iii)the amount of two or more antibodies which bind to said two or moredifferent tumor-associated antigens or to parts thereof, and/or (iv) theamount of two or more cytolytic T cells or of T helper cells which arespecific for complexes between said two or more differenttumor-associated antigens or of parts thereof and MHC molecules.

According to the present technology, detection of a nucleic acid or of apart thereof or monitoring the amount of a nucleic acid or of a partthereof may be carried out using a polynucleotide probe which hybridizesspecifically to said nucleic acid or said part thereof or may be carriedout by selective amplification of said nucleic acid or said partthereof. In one embodiment, the polynucleotide probe comprises asequence of 6-50, in particular 10-30, 15-30 and 20-30, contiguousnucleotides of said nucleic acid.

In particular embodiments, the tumor-associated antigen to be detectedor the part thereof is present intracellularly or on the cell surface.According to the present technology, detection of a tumor-associatedantigen or of a part thereof or monitoring the amount of atumor-associated antigen or of a part thereof may be carried out usingan antibody binding specifically to said tumor-associated antigen orsaid part thereof.

In further embodiments, the tumor-associated antigen to be detected orthe part thereof is present in a complex with an MHC molecule, inparticular an HLA molecule.

According to the present technology, detection of an antibody ormonitoring the amount of antibodies may be carried out using a proteinor peptide binding specifically to said antibody.

According to the present technology, detection of cytolytic T cells orof T helper cells or monitoring the amount of cytolytic T cells or of Thelper cells which are specific for complexes between an antigen or apart thereof and MHC molecules may be carried out using a cellpresenting the complex between said antigen or said part thereof and anMHC molecule.

The polynucleotide probe, the antibody, the protein or peptide or thecell, which is used for detection or monitoring, is preferably labeledin a detectable manner. In particular embodiments, the detectable markeris a radioactive marker or an enzymic marker. T lymphocytes mayadditionally be detected by detecting their proliferation, theircytokine production, and their cytotoxic activity triggered by specificstimulation with the complex of MHC and tumor-associated antigen orparts thereof. T lymphocytes may also be detected via a recombinant MHCmolecule or else a complex of two or more MHC molecules which are loadedwith the particular immunogenic fragment of one or more of thetumor-associated antigens and which can identify the specific Tlymphocytes by contacting the specific T cell receptor.

In a further aspect, the present technology relates to a method oftreating, diagnosing or monitoring a disease characterized by expressionor abnormal expression of a tumor-associated antigen identifiedaccording to the present technology, which method comprisesadministering an antibody which binds to said tumor-associated antigenor to a part thereof and which is coupled to a therapeutic or diagnosticagent. The antibody may be a monoclonal antibody. In furtherembodiments, the antibody is a chimeric or humanized antibody or afragment of a natural antibody.

The present technology also relates to a method of treating a patienthaving a disease characterized by expression or abnormal expression of atumor-associated antigen identified according to the present technology,which method comprises (i) removing a sample containing immunoreactivecells from said patient, (ii) contacting said sample with a host cellexpressing said tumor-associated antigen or a part thereof, underconditions which favor production of cytolytic T cells against saidtumor-associated antigen or a part thereof, and (iii) introducing thecytolytic T cells into the patient in an amount suitable for lysingcells expressing the tumor-associated antigen or a part thereof. Thepresent technology likewise relates to cloning the T cell receptor ofcytolytic T cells against the tumor-associated antigen. Said receptormay be transferred to other T cells which thus receive the desiredspecificity and, as under (iii), may be introduced into the patient.

In one embodiment, the host cell endogenously expresses an HLA molecule.In a further embodiment, the host cell recombinantly expresses an HLAmolecule and/or the tumor-associated antigen or the part thereof. Thehost cell is preferably nonproliferative. In a preferred embodiment, thehost cell is an antigen-presenting cell, in particular a dendritic cell,a monocyte or a macrophage.

In a further aspect, the present technology relates to a method oftreating a patient having a disease characterized by expression orabnormal expression of a tumor-associated antigen, which methodcomprises (i) identifying a nucleic acid which codes for atumor-associated antigen identified according to the present technologyand which is expressed by cells associated with said disease, (ii)transfecting a host cell with said nucleic acid or a part thereof, (iii)culturing the transfected host cell for expression of said nucleic acid(this is not obligatory when a high rate of transfection is obtained),and (iv) introducing the host cells or an extract thereof into thepatient in an amount suitable for increasing the immune response to thepatient's cells associated with the disease. The method may furthercomprise identifying an MHC molecule presenting the tumor-associatedantigen or a part thereof, with the host cell expressing the identifiedMHC molecule and presenting said tumor-associated antigen or a partthereof. The immune response may comprise a B cell response or a T cellresponse. Furthermore, a T cell response may comprise production ofcytolytic T cells and/or T helper cells which are specific for the hostcells presenting the tumor-associated antigen or a part thereof orspecific for cells of the patient which express said tumor-associatedantigen or a part thereof.

The present technology also relates to a method of treating a diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen identified according to the present technology, which methodcomprises (i) identifying cells from the patient which express abnormalamounts of the tumor-associated antigen, (ii) isolating a sample of saidcells, (iii) culturing said cells, and (iv) introducing said cells intothe patient in an amount suitable for triggering an immune response tothe cells.

Preferably, the host cells used according to the present technology arenonproliferative or are rendered nonproliferative. A diseasecharacterized by expression or abnormal expression of a tumor-associatedantigen is in particular cancer.

The present technology furthermore relates to a nucleic acid selectedfrom the group consisting of (a) a nucleic acid which comprises anucleic acid sequence selected from the group consisting of SEQ ID NOs:3-5, a part or derivative thereof, (b) a nucleic acid which hybridizeswith the nucleic acid of (a) under stringent conditions, (c) a nucleicacid which is degenerate with respect to the nucleic acid of (a) or (b),and (d) a nucleic acid which is complementary to the nucleic acid of(a), (b) or (c). The present technology furthermore relates to a nucleicacid, which codes for a protein or polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 10 and 12-14,a part or derivative thereof.

In a further aspect, the present technology relates to promotersequences of nucleic acids of the present technology. These sequencesmay be functionally linked to another gene, preferably in an expressionvector, and thus ensure selective expression of said gene in appropriatecells.

In a further aspect, the present technology relates to a recombinantnucleic acid molecule, in particular DNA or RNA molecule, whichcomprises a nucleic acid of the present technology.

The present technology also relates to host cells which contain anucleic acid of the present technology or a recombinant nucleic acidmolecule comprising a nucleic acid of the present technology.

The host cell may also comprise a nucleic acid coding for a HLAmolecule. In one embodiment, the host cell endogenously expresses theHLA molecule. In a further embodiment, the host cell recombinantlyexpresses the HLA molecule and/or the nucleic acid of the presenttechnology or a part thereof. Preferably, the host cell isnonproliferative. In a preferred embodiment, the host cell is anantigen-presenting cell, in particular a dendritic cell, a monocyte or amacrophage.

In a further embodiment, the present technology relates tooligonucleotides which hybridize with a nucleic acid identifiedaccording to the present technology and which may be used as geneticprobes or as “antisense” molecules. Nucleic acid molecules in the formof oligonucleotide primers or competent samples, which hybridize with anucleic acid identified according to the present technology or partsthereof, may be used for finding nucleic acids which are homologous tosaid nucleic acid identified according to the present technology. PCRamplification, Southern and Northern hybridization may be employed forfinding homologous nucleic acids. Hybridization may be carried out underlow stringency, more preferably under medium stringency and mostpreferably under high stringency conditions. The term “stringentconditions” according to the present technology refers to conditionswhich allow specific hybridization between polynucleotides.

In a further aspect, the present technology relates to a protein,polypeptide or peptide which is encoded by a nucleic acid selected fromthe group consisting of (a) a nucleic acid which comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 3-5, apart or derivative thereof, (b) a nucleic acid which hybridizes with thenucleic acid of (a) under stringent conditions, (c) a nucleic acid whichis degenerate with respect to the nucleic acid of (a) or (b), and (d) anucleic acid which is complementary to the nucleic acid of (a), (b) or(c). In a preferred embodiment, the present technology relates to aprotein or polypeptide or peptide which comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 10 and 12-14, a partor derivative thereof.

In a further aspect, the present technology relates to an immunogenicfragment of a tumor-associated antigen identified according to thepresent technology. Said fragment preferably binds to a human HLAreceptor or to a human antibody. A fragment of the present technologypreferably comprises a sequence of at least 6, in particular at least 8,at least 10, at least 12, at least 15, at least 20, at least 30 or atleast 50, amino acids.

In this aspect the present technology relates, in particular, to apeptide which has or comprises a sequence selected from the groupconsisting of SEQ ID NOs: 17-19, 90-97, 100-102, 105, 106, 111-116, 120,123, 124, and 135-137, a part or derivative thereof.

In a further aspect, the present technology relates to an agent whichbinds to a tumor-associated antigen identified according to the presenttechnology or to a part thereof. In a preferred embodiment, the agent isan antibody. In further embodiments, the antibody is a chimeric, ahumanized antibody or an antibody produced by combinatory techniques oris a fragment of an antibody. Furthermore, the present technologyrelates to an antibody which binds selectively to a complex of (i) atumor-associated antigen identified according to the present technologyor a part thereof and (ii) an MHC molecule to which saidtumor-associated antigen identified according to the present technologyor said part thereof binds, with said antibody not binding to (i) or(ii) alone. An antibody of the present technology may be a monoclonalantibody. In further embodiments, the antibody is a chimeric orhumanized antibody or a fragment of a natural antibody.

In particular, the present technology relates to such an agent, inparticular an antibody, which specifically binds to a peptide which hasor comprises a sequence selected from the group consisting of SEQ IDNOs: 17-19, 90-97, 100-102, 105, 106, 111-116, 120, 123, 124, and135-137, a part or derivative thereof.

The present technology furthermore relates to a conjugate between anagent of the present technology which binds to a tumor-associatedantigen identified according to the present technology or to a partthereof or an antibody of the present technology and a therapeutic ordiagnostic agent. In one embodiment, the therapeutic or diagnostic agentis a toxin.

In a further aspect, the present technology relates to a kit fordetecting expression or abnormal expression of a tumor-associatedantigen identified according to the present technology, which kitcomprises agents for detection (i) of the nucleic acid which codes forthe tumor-associated antigen or of a part thereof, (ii) of thetumor-associated antigen or of a part thereof, (iii) of antibodies whichbind to the tumor-associated antigen or to a part thereof, and/or (iv)of T cells which are specific for a complex between the tumor-associatedantigen or a part thereof and an MHC molecule. In one embodiment, theagents for detection of the nucleic acid or the part thereof are nucleicacid molecules for selective amplification of said nucleic acid, whichcomprise, in particular a sequence of 6-50, in particular 10-30, 15-30and 20-30, contiguous nucleotides of said nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

According to the present technology, genes are described which areexpressed in tumor cells selectively or aberrantly and which aretumor-associated antigens.

According to the present technology, these genes and/or their geneticproducts and/or their derivatives and/or parts are preferred targetstructures for therapeutic approaches. Conceptionally, said therapeuticapproaches may aim at inhibiting the activity of the selectivelyexpressed tumor-associated genetic product. This is useful, if saidaberrant respective selective expression is functionally important intumor pathogenecity and if its ligation is accompanied by selectivedamage of the corresponding cells. Other therapeutic conceptscontemplate tumor-associated antigens as labels which recruit effectormechanisms having cell-damaging potential selectively to tumor cells.Here, the function of the target molecule itself and its role in tumordevelopment are totally irrelevant.

“Derivative” of a nucleic acid means according to the present technologythat single or multiple nucleotide substitutions, deletions and/oradditions are present in said nucleic acid. Furthermore, the term“derivative” also comprises chemical derivatization of a nucleic acid ona nucleotide base, on the sugar or on the phosphate. The term“derivative” also comprises nucleic acids which contain nucleotides andnucleotide analogs not occurring naturally.

According to the present technology, a nucleic acid is preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acidscomprise according to the present technology genomic DNA, cDNA, mRNA,recombinantly produced and chemically synthesized molecules. Accordingto the present technology, a nucleic acid may be present as asingle-stranded or double-stranded and linear or covalently circularlyclosed molecule.

The nucleic acids described according to the present technology havepreferably been isolated. The term “isolated nucleic acid” meansaccording to the present technology that the nucleic acid was (i)amplified in vitro, for example by polymerase chain reaction (PCR), (ii)recombinantly produced by cloning, (iii) purified, for example bycleavage and gel-electrophoretic fractionation, or (iv) synthesized, forexample by chemical synthesis. An isolated nucleic acid is a nucleicacid which is available for manipulation by recombinant DNA techniques.

A nucleic acid is “complementary” to another nucleic acid if the twosequences are capable of hybridizing and forming a stable duplex withone another, with hybridization preferably being carried out underconditions which allow specific hybridization between polynucleotides(stringent conditions). Stringent conditions are described, for example,in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989 or Current Protocols in Molecular Biology, F. M. Ausubel etal., Editors, John Wiley & Sons, Inc., New York and refer, for example,to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mMNaH₂PO₄ (pH 7), 0.5% SOS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15M sodium citrate, pH 7. After hybridization, the membrane to which theDNA has been transferred is washed, for example, in 2×SSC at roomtemperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68°C.

According to the present technology, complementary nucleic acids have atleast 40%, in particular at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% and preferably at least 95%, at least 98% or atleast 99%, identical nucleotides.

Nucleic acids coding for tumor-associated antigens may, according to thepresent technology, be present alone or in combination with othernucleic acids, in particular heterologous nucleic acids. In preferredembodiments, a nucleic acid is functionally linked to expression controlsequences or regulatory sequences which may be homologous orheterologous with respect to said nucleic acid. A coding sequence and aregulatory sequence are “functionally” linked to one another, if theyare covalently linked to one another in such a way that expression ortranscription of said coding sequence is under the control or under theinfluence of said regulatory sequence. If the coding sequence is to betranslated into a functional protein, then, with a regulatory sequencefunctionally linked to said coding sequence, induction of saidregulatory sequence results in transcription of said coding sequence,without causing a frame shift in the coding sequence or said codingsequence not being capable of being translated into the desired proteinor peptide.

The term “expression control sequence” or “regulatory sequence”comprises according to the present technology promoters, enhancers andother control elements which regulate expression of a gene. Inparticular embodiments of the present technology, the expression controlsequences can be regulated. The exact structure of regulatory sequencesmay vary as a function of the species or cell type, but generallycomprises 5′ untranscribed and 5′ untranslated sequences which areinvolved in initiation of transcription and translation, respectively,such as TATA box, capping sequence, CAAT sequence, and the like. Morespecifically, 5′ untranscribed regulatory sequences comprise a promoterregion which includes a promoter sequence for transcriptional control ofthe functionally linked gene. Regulatory sequences may also compriseenhancer sequences or upstream activator sequences.

Thus, on the one hand, the tumor-associated antigens illustrated hereinmay be combined with any expression control sequences and promoters. Onthe other hand, however, the promoters of the tumor-associated geneticproducts illustrated herein may, according to the present technology, becombined with any other genes. This allows the selective activity ofthese promoters to be utilized.

According to the present technology, a nucleic acid may furthermore bepresent in combination with another nucleic acid which codes for apolypeptide controlling secretion of the protein or polypeptide encodedby said nucleic acid from a host cell. According to the presenttechnology, a nucleic acid may also be present in combination withanother nucleic acid which codes for a polypeptide causing the encodedprotein or polypeptide to be anchored on the cell membrane of the hostcell or compartmentalized into particular organelles of said cell.Similarly, a combination with a nucleic acid is possible whichrepresents a reporter gene or any “tag”.

In a preferred embodiment, a recombinant DNA molecule is according tothe present technology a vector, where appropriate with a promoter,which controls expression of a nucleic acid, for example a nucleic acidcoding for a tumor-associated antigen of the present technology. Theterm “vector” is used here in its most general meaning and comprises anyintermediary vehicle for a nucleic acid which enables said nucleic acid,for example, to be introduced into prokaryotic and/or eukaryotic cellsand, where appropriate, to be integrated into a genome. Vectors of thiskind are preferably replicated and/or expressed in the cells. Anintermediary vehicle may be adapted, for example, to the use inelectroporation, in bombardment with microprojectiles, in liposomaladministration, in the transfer with the aid of agrobacteria or ininsertion via DNA or RNA viruses. Vectors comprise plasmids, phagemidsor viral genomes.

The nucleic acids coding for a tumor-associated antigen identifiedaccording to the present technology may be used for transfection of hostcells. Nucleic acids here mean both recombinant DNA and RNA. RecombinantRNA may be prepared by in-vitro transcription of a DNA template.Furthermore, it may be modified by stabilizing sequences, capping andpolyadenylation prior to application.

According to the present technology, the term “host cell” relates to anycell which can be transformed or transfected with an exogenous nucleicacid. The term “host cells” comprises according to the presenttechnology prokaryotic (e.g. E. coli) or eukaryotic cells (e.g.dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast cellsand insect cells). Particular preference is given to mammalian cellssuch as cells from humans, mice, hamsters, pigs, goats, primates. Thecells may be derived from a multiplicity of tissue types and compriseprimary cells and cell lines.

Specific examples comprise keratinocytes, peripheral blood leukocytes,stem cells of the bone marrow and embryonic stem cells. In furtherembodiments, the host cell is an antigen-presenting cell, in particulara dendritic cell, monocyte or a macrophage. A nucleic acid may bepresent in the host cell in the form of a single copy or of two or morecopies and, in one embodiment, is expressed in the host cell.

According to the present technology, the term “expression” is used inits most general meaning and comprises the production of RNA or of RNAand protein. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.Preferred expression systems in mammalian cells comprise pcDNA3.1 andpRc/CMV (Invitrogen, Carlsbad, Calif.), which contain a selectablemarker such as a gene imparting resistance to G418 (and thus enablingstably transfected cell lines to be selected) and the enhancer-promotersequences of cytomegalovirus (CMV).

In those cases of the present technology in which an HLA moleculepresents a tumor-associated antigen or a part thereof, an expressionvector may also comprise a nucleic acid sequence coding for said HLAmolecule. The nucleic acid sequence coding for the HLA molecule may bepresent on the same expression vector as the nucleic acid coding for thetumor-associated antigen or the part thereof, or both nucleic acids maybe present on different expression vectors. In the latter case, the twoexpression vectors may be cotransfected into a cell. If a host cellexpresses neither the tumor-associated antigen or the part thereof northe HLA molecule, both nucleic acids coding therefor are transfectedinto the cell either on the same expression vector or on differentexpression vectors. If the cell already expresses the HLA molecule, onlythe nucleic acid sequence coding for the tumor-associated antigen or thepart thereof can be transfected into the cell.

The present technology also comprises kits for amplification of anucleic acid coding for a tumor-associated antigen. Such kits comprise,for example, a pair of amplification primers which hybridize to thenucleic acid coding for the tumor-associated antigen. The primerspreferably comprise a sequence of 6-50, in particular 10-30, 15-30 and20-30 contiguous nucleotides of the nucleic acid and are nonoverlapping,in order to avoid the formation of primer dimers. One of the primerswill hybridize to one strand of the nucleic acid coding for thetumor-associated antigen, and the other primer will hybridize to thecomplementary strand in an arrangement which allows amplification of thenucleic acid coding for the tumor-associated antigen.

“Antisense” molecules or “antisense” nucleic acids may be used forregulating, in particular reducing, expression of a nucleic acid. Theterm “antisense molecule” or “antisense nucleic acid” refers accordingto the present technology to an oligonucleotide which is anoligoribonucleotide, oligodeoxyribonucleotide, modifiedoligoribonucleotide or modified oligodeoxyribonucleotide and whichhybridizes under physiological conditions to DNA comprising a particulargene or to mRNA of said gene, thereby inhibiting transcription of saidgene and/or translation of said mRNA. According to the presenttechnology, an “antisense molecule” also comprises a construct whichcontains a nucleic acid or a part thereof in reverse orientation withrespect to its natural promoter. An antisense transcript of a nucleicacid or of a part thereof may form a duplex with the naturally occurringmRNA specifying the enzyme and thus prevent accumulation of ortranslation of the mRNA into the active enzyme.

Another possibility is the use of ribozymes for inactivating a nucleicacid. Antisense oligonucleotides preferred according to the presenttechnology have a sequence of 6-50, in particular 10-30, 15-30 and20-30, contiguous nucleotides of the target nucleic acid and preferablyare fully complementary to the target nucleic acid or to a part thereof.

In preferred embodiments, the antisense oligonucleotide hybridizes withan N-terminal or 5′ upstream site such as a translation initiation site,transcription initiation site or promoter site. In further embodiments,the antisense oligonucleotide hybridizes with a 3′ untranslated regionor mRNA splicing site.

In one embodiment, an oligonucleotide of the present technology consistsof ribonucleotides, deoxyribonucleotides or a combination thereof, withthe 5′ end of one nucleotide and the 3′ end of another nucleotide beinglinked to one another by a phosphodiester bond. These oligonucleotidesmay be synthesized in the conventional manner or produced recombinantly.

In preferred embodiments, an oligonucleotide of the present technologyis a “modified” oligonucleotide. Here, the oligonucleotide may bemodified in very different ways, without impairing its ability to bindits target, in order to increase, for example, its stability ortherapeutic efficacy. According to the present technology, the term“modified oligonucleotide” means an oligonucleotide in which (i) atleast two of its nucleotides are linked to one another by a syntheticinternucleoside bond (i.e. an internucleoside bond which is not aphosphodiester bond) and/or (ii) a chemical group which is usually notfound in nucleic acids is covalently linked to the oligonucleotide.Preferred synthetic internucleoside bonds are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphatetriesters, acetamidates, carboxymethyl esters and peptides.

The term “modified oligonucleotide” also comprises oligonucleotideshaving a covalently modified base and/or sugar. “Modifiedoligonucleotides” comprise, for example, oligonucleotides with sugarresidues which are covalently bound to low molecular weight organicgroups other than a hydroxyl group at the 3′ position and a phosphategroup at the 5′ position. Modified oligonucleotides may comprise, forexample, a 2′-O-alkylated ribose residue or another sugar instead ofribose, such as arabinose.

Preferably, the proteins and polypeptides described according to thepresent technology have been isolated. The terms “isolated protein” or“isolated polypeptide” mean that the protein or polypeptide has beenseparated from its natural environment. An isolated protein orpolypeptide may be in an essentially purified state. The term“essentially purified” means that the protein or polypeptide isessentially free of other substances with which it is associated innature or in vivo.

Such proteins and polypeptides may be used, for example, in producingantibodies and in an immunological or diagnostic assay or astherapeutics. Proteins and polypeptides described according to thepresent technology may be isolated from biological samples such astissue or cell homogenates and may also be expressed recombinantly in amultiplicity of pro- or eukaryotic expression systems.

For the purposes of the present technology, “derivatives” of a proteinor polypeptide or of an amino acid sequence comprise amino acidinsertion variants, amino acid deletion variants and/or amino acidsubstitution variants.

Amino acid insertion variants comprise amino- and/or carboxy-terminalfusions and also insertions of single or two or more amino acids in aparticular amino acid sequence. In the case of amino acid sequencevariants having an insertion, one or more amino acid residues areinserted into a particular site in an amino acid sequence, althoughrandom insertion with appropriate screening of the resulting product isalso possible. Amino acid deletion variants are characterized by theremoval of one or more amino acids from the sequence. Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpolypeptides. Preference is given to replacing amino acids with otherones having similar properties such as hydrophobicity, hydrophilicity,electronegativity, volume of the side chain and the like (conservativesubstitution). Conservative substitutions, for example, relate to theexchange of one amino acid with another amino acid listed below in thesame group as the amino acid to be substituted:

1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly)

2. negatively charged residues and their amides: Asn, Asp, Glu, Gin

3. positively charged residues: His, Arg, Lys

4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys)

5. large aromatic residues: Phe, Tyr, Trp.

Owing to their particular part in protein architecture, three residuesare shown in brackets. Gly is the only residue without a side chain andthus imparts flexibility to the chain. Pro has an unusual geometry whichgreatly restricts the chain. Cys can form a disulfide bridge.

The amino acid variants described above may be readily prepared with theaid of known peptide synthesis techniques such as, for example, by solidphase synthesis (Merrifield, 1964) and similar methods or by recombinantDNA manipulation. Techniques for introducing substitution mutations atpredetermined sites into DNA which has a known or partially knownsequence are well known and comprise M13 mutagenesis, for example. Themanipulation of DNA sequences for preparing proteins havingsubstitutions, insertions or deletions, is described in detail inSambrook et al. (1989), for example.

According to the present technology, “derivatives” of proteins,polypeptides or peptides also comprise single or multiple substitutions,deletions and/or additions of any molecules associated with the enzyme,such as carbohydrates, lipids and/or proteins, polypeptides or peptides.The term “derivative” also extends to all functional chemicalequivalents of said proteins, polypeptides or peptides.

According to the present technology, a part or fragment of atumor-associated antigen has a functional property of the polypeptidefrom which it has been derived. Such functional properties comprise theinteraction with antibodies, the interaction with other polypeptides orproteins, the selective binding of nucleic acids and an enzymaticactivity. A particular property is the ability to form a complex withHLA and, where appropriate, generate an immune response. This immuneresponse may be based on stimulating cytotoxic or T helper cells. A partor fragment of a tumor-associated antigen of the present technologypreferably comprises a sequence of at least 6, in particular at least 8,at least 10, at least 12, at least 15, at least 20, at least 30 or atleast 50, consecutive amino acids of the tumor-associated antigen.

A part or a fragment of a nucleic acid coding for a tumor-associatedantigen relates according to the present technology to the part of thenucleic acid, which codes at least for the tumor-associated antigenand/or for a part or a fragment of said tumor-associated antigen, asdefined above.

The isolation and identification of genes coding for tumor-associatedantigens also make possible the diagnosis of a disease characterized byexpression of one or more tumor-associated antigens. These methodscomprise determining one or more nucleic acids which code for atumor-associated antigen and/or determining the encoded tumor-associatedantigens and/or peptides derived therefrom. The nucleic acids may bedetermined in the conventional manner, including by polymerase chainreaction or hybridization with a labeled probe. Tumor-associatedantigens or peptides derived therefrom may be determined by screeningpatient antisera with respect to recognizing the antigen and/or thepeptides. They may also be determined by screening T cells of thepatient for specificities for the corresponding tumor-associatedantigen.

The present technology also enables proteins binding to tumor-associatedantigens described herein to be isolated, including antibodies andcellular binding partners of said tumor-associated antigens.

According to the present technology, particular embodiments ought toinvolve providing “dominant negative” polypeptides derived fromtumor-associated antigens. A dominant negative polypeptide is aninactive protein variant which, by way of interacting with the cellularmachinery, displaces an active protein from its interaction with thecellular machinery or which competes with the active protein, therebyreducing the effect of said active protein. For example, a dominantnegative receptor which binds to a ligand but does not generate anysignal as response to binding to the ligand can reduce the biologicaleffect of said ligand. Similarly, a dominant negative catalyticallyinactive kinase which usually interacts with target proteins but doesnot phosphorylate said target proteins may reduce phosphorylation ofsaid target proteins as response to a cellular signal. Similarly, adominant negative transcription factor which binds to a promoter site inthe control region of a gene but does not increase transcription of saidgene may reduce the effect of a normal transcription factor by occupyingpromoter binding sites, without increasing transcription.

The result of expression of a dominant negative polypeptide in a cell isa reduction in the function of active proteins. The skilled worker mayprepare dominant negative variants of a protein, for example, byconventional mutagenesis methods and by evaluating the dominant negativeeffect of the variant polypeptide.

The present technology also comprises substances such as polypeptideswhich bind to tumor-associated antigens. Such binding substances may beused, for example, in screening assays for detecting tumor-associatedantigens and complexes of tumor-associated antigens with their bindingpartners and in the purification of said tumor-associated antigens andof complexes thereof with their binding partners. Such substances mayalso be used for inhibiting the activity of tumor-associated dominantantigens, for example by binding to such antigens.

The present technology therefore comprises binding substances such as,for example, antibodies or antibody fragments, which are capable ofselectively binding to tumor-associated antigens. Antibodies comprisepolyclonal and monoclonal antibodies which are produced in theconventional manner.

Such antibodies can recognize proteins in the native and/or denaturatedstate (Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll, J.Immunol. Methods 234: 107-116, 2000; Kayyem et al., Eur. J. Biochem.208: 1-8, 1992; Spiller et al., J. Immunol. Methods 224: 51-60, 1999).

Antisera which contain specific antibodies specifically binding to thetarget protein can be prepared by various standard processes; see, forexample, “Monoclonal Antibodies: A Practical Approach” by PhilipShepherd, Christopher Dean ISBN 0-19-963722-9; “Antibodies: A LaboratoryManual” by Ed Harlow, David Lane, ISBN: 0879693142 and “UsingAntibodies: A Laboratory Manual: Portable Protocol NO” by Edward Harlow,David Lane, Ed Harlow ISBN 0879695447. Thereby it is also possible togenerate affine and specific antibodies which recognize complex membraneproteins in their native form (Azorsa et al., J. Immunol. Methods 229:35-48, 1999; Anderson et al., J Immunol. 143: 30 1899-1904, 1989;Gardsvoll, J Immunol. Methods 234: 107-116, 2000). This is in particularrelevant for the preparation of antibodies which are to be usedtherapeutically, but also for many diagnostic applications. In thisrespect, it is possible to immunize with the whole protein, withextracellular partial sequences as well as with cells which express thetarget molecule in physiologically folded form.

Monoclonal antibodies are traditionally prepared using the hybridomatechnology. (for technical details see: “Monoclonal Antibodies: APractical Approach” by Philip Shepherd, Christopher Dean ISBN0-19-963722-9; “Antibodies: A Laboratory Manual” by Ed Harlow, DavidLane ISBN: 0879693142; “Using Antibodies: A Laboratory Manual: PortableProtocol NO” by Edward Harlow, David Lane, Ed Harlow ISBN: 0879695447).

It is known that only a small part of an antibody molecule, theparatope, is involved in binding of the antibody to its epitope (cf.Clark, W. R. (1986), The Experimental Foundations of Modern Immunology,Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology,7th Edition, Blackwell Scientific Publications, Oxford). The pFc′ and Fcregions are, for example, effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically removed or which has been produced without thepFc′ region, referred to as F(ab′)₂ fragment, carries both antigenbinding sites of a complete antibody. Similarly, an antibody from whichthe Fc region has been enzymatically removed or which has been producedwithout said Fc region, referred to as Fab fragment, carries one antigenbinding site of an intact antibody molecule. Furthermore, Fab fragmentsconsist of a covalently bound light chain of an antibody and part of theheavy chain of said antibody, referred to as Fd. The Fd fragments arethe main determinants of antibody specificity (a single Fd fragment canbe associated with up to ten different light chains, without alteringthe specificity of the antibody) and Fd fragments, when isolated, retainthe ability to bind to an epitope.

Located within the antigen-binding part of an antibody arecomplementary-determining regions (CDRs) which interact directly withthe antigen epitope and framework regions (FRs) which maintain thetertiary structure of the paratope. Both the Fd fragment of the heavychain and the light chain of IgG immunoglobulins contain four frameworkregions (FR1 to FR4) which are separated in each case by threecomplementary-determining regions (CDR1 to CDR3). The CDRs and, inparticular, the CDR3 regions and, still more particularly, the CDR3region of the heavy chain are responsible to a large extent for antibodyspecificity.

Non-CDR regions of a mammalian antibody are known to be able to bereplaced by similar regions of antibodies with the same or a differentspecificity, with the specificity for the epitope of the originalantibody being retained. This made possible the development of“humanized” antibodies in which nonhuman CDRs are covalently linked tohuman FR and/or Fc/pFc′ regions to produce a functional antibody.

This is utilized in the so called “SLAM” technology, wherein B cellsfrom whole blood are isolated and the cells are monocloned. Then, thesupernatant of the single B cells is analyzed with respect to itsantibody specificity. In contrast to the hybridoma technology thevariable region of the antibody gene is amplified using single cell PCRand cloned into a suitable vector. In this way, the provision ofmonoclonal antibodies is accelerated (de Wildt et al., J. Immunol.Methods 207: 61-67, 1997).

As another example, WO 92/04381 describes the production and use ofhumanized murine RSV antibodies in which at least part of the murine FRregions have been replaced with FR regions of a human origin. Antibodiesof this kind, including fragments of intact antibodies withantigen-binding capability, are often referred to as “chimeric”antibodies.

The present technology also provides F(ab′)₂, Fab, Fv, and Fd fragmentsof antibodies, chimeric antibodies, in which the Fc and/or FR and/orCDR1 and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, chimeric F(ab′)₂-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain-CDR3 regions have been replaced with homologous human or nonhumansequences, chimeric Fab-fragment antibodies in which the FR and/or CDR1and/or CDR2 and/or light chain-CDR3 regions have been replaced withhomologous human or nonhuman sequences, and chimeric Fd-fragmentantibodies in which the FR and/or CDR1 and/or CDR2 regions have beenreplaced with homologous human or nonhuman sequences. The presenttechnology also comprises “single-chain” antibodies.

The present technology also comprises polypeptides which bindspecifically to tumor-associated antigens. Polypeptide bindingsubstances of this kind may be provided, for example, by degeneratepeptide libraries which may be prepared simply in solution in animmobilized form or as phage-display libraries. It is likewise possibleto prepare combinatorial libraries of peptides with one or more aminoacids. Libraries of peptoids and nonpeptidic synthetic residues may alsobe prepared.

Phage display may be particularly effective in identifying bindingpeptides of the present technology. In this connection, for example, aphage library is prepared (using, for example, the M13, fd or lambdaphages) which presents inserts of from 4 to about 80 amino acid residuesin length. Phages are then selected which carry inserts which bind tothe tumor-associated antigen. This process may be repeated via two ormore cycles of a reselection of phages binding to the tumor-associatedantigen. Repeated rounds result in a concentration of phages carryingparticular sequences. An analysis of DNA sequences may be carried out inorder to identify the sequences of the expressed polypeptides. Thesmallest linear portion of the sequence binding to the tumor-associatedantigen may be determined. The “two-hybrid system” of yeast may also beused for identifying polypeptides which bind to a tumor-associatedantigen. Tumor-associated antigens described according to the presenttechnology or fragments thereof may be used for screening peptidelibraries, including phage-display libraries, in order to identify andselect peptide binding partners of the tumor-associated antigens. Suchmolecules may be used, for example, for screening assays, purificationprotocols, for interference with the function of the tumor-associatedantigen and for other purposes known to the skilled worker.

The antibodies described above and other binding molecules may be used,for example, for identifying tissue which expresses a tumor-associatedantigen. Antibodies may also be coupled to specific diagnosticsubstances for displaying cells and tissues expressing tumor-associatedantigens. They may also be coupled to therapeutically useful substances.Diagnostic substances comprise, in a nonlimiting manner, barium sulfate,iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate,meglumine diatrizoate, metrizamide, sodium tyropanoate and radiodiagnostic, including positron emitters such as fluorine-18 andcarbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131and indium-111, nuclides for nuclear magnetic resonance, such asfluorine and gadolinium. According to the present technology, the term“therapeutically useful substance” means any therapeutic molecule which,as desired, is selectively guided to a cell which expresses one or moretumor-associated antigens, including anticancer agents, radioactiveiodine-labeled compounds, toxins, cytostatic or cytolytic drugs, etc.Anticancer agents comprise, for example, aminoglutethimide,azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil,cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine,dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil,interferon-α, lomustine, mercaptopurine, methotrexate, mitotane,procarbazine HCl, thioguanine, vinblastine sulfate and vincristinesulfate. Other anticancer agents are described, for example, in Goodmanand Gilman, “The Pharmacological Basis of Therapeutics”, 8th Edition,1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic Agents(Paul Calabresi and Bruce A. Chabner). Toxins may be proteins such aspokeweed antiviral protein, cholera toxin, pertussis toxin, ricin,gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin. Toxinresidues may also be high energy-emitting radionuclides such ascobalt-60.

The term “patient” means according to the present technology a humanbeing, a nonhuman primate or another animal, in particular a mammal suchas a cow, horse, pig, sheep, goat, dog, cat or a rodent such as a mouseand rat. In a particularly preferred embodiment, the patient is a humanbeing.

According to the present technology, the term “disease” refers to anypathological state in which tumor-associated antigens are expressed orabnormally expressed. “Abnormal expression” means according to thepresent technology that expression is altered, preferably increased,compared to the state in a healthy individual. An increase in expressionrefers to an increase by at least 10%, in particular at least 20%, atleast 50% or at least 100%. In one embodiment, the tumor-associatedantigen is expressed only in tissue of a diseased individual, whileexpression in a healthy individual is repressed. One example of such adisease is cancer, wherein the term “cancer” according to the presenttechnology comprises leukemias, seminomas, melanomas, teratomas,gliomas, kidney cancer, adrenal cancer, thyroid cancer, intestinalcancer, liver cancer, colon cancer, stomach cancer, gastrointestinalcancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreascancer, ear, nose and throat (ENT) cancer, breast cancer, prostatecancer, cancer of the uterus, ovarian cancer and lung cancer and thematastases thereof.

According to the present technology, a biological sample may be a tissuesample and/or a cellular sample and may be obtained in the conventionalmanner such as by tissue biopsy, including punch biopsy, and by takingblood, bronchial aspirate, sputum, urine, feces or other body fluids,for use in the various methods described herein.

According to the present technology, the term “immunoreactive cell”means a cell which can mature into an immune cell (such as B cell, Thelper cell, or cytolytic T cell) with suitable stimulation.Immunoreactive cells comprise CD34⁺ hematopoietic stem cells, immatureand mature T cells and immature and mature B cells. If production ofcytolytic or T helper cells recognizing a tumor-associated antigen isdesired, the immunoreactive cell is contacted with a cell expressing atumor-associated antigen under conditions which favor production,differentiation and/or selection of cytolytic T cells and of T helpercells. The differentiation of T cell precursors into a cytolytic T cell,when exposed to an antigen, is similar to clonal selection of the immunesystem.

Some therapeutic methods are based on a reaction of the immune system ofa patient, which results in a lysis of antigen-presenting cells such ascancer cells which present one or more tumor-associated antigens. Inthis connection, for example autologous cytotoxic T lymphocytes specificfor a complex of a tumor-associated antigen and an MHC molecule areadministered to a patient having a cellular abnormality. The productionof such cytotoxic T lymphocytes in vitro is known. An example of amethod of differentiating T cells can be found in WO-A-9633265.Generally, a sample containing cells such as blood cells is taken fromthe patient and the cells are contacted with a cell which presents thecomplex and which can cause propagation of cytotoxic T lymphocytes (e.g.dendritic cells). The target cell may be a transfected cell such as aCOS cell. These transfected cells present the desired complex on theirsurface and, when contacted with cytotoxic T lymphocytes, stimulatepropagation of the latter. The clonally expanded autologous cytotoxic Tlymphocytes are then administered to the patient.

In another method of selecting antigen-specific cytotoxic T lymphocytes,fluorogenic tetramers of MHC class I molecule/peptide complexes are usedfor detecting specific clones of cytotoxic T lymphocytes (Altman et al.,Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998).Soluble MHC class I molecules are folded in vitro in the presence of β₂microglobulin and a peptide antigen binding to said class I molecule.The MHC/peptide complexes are purified and then labeled with biotin.Tetramers are formed by mixing the biotinylated peptide-MHC complexeswith labeled avidin (e.g. phycoerythrin) in a molar ratio of 4:1.Tetramers are then contacted with cytotoxic T lymphocytes such asperipheral blood or lymph nodes. The tetramers bind to cytotoxic Tlymphocytes which recognize the peptide antigen/MHC class I complex.Cells which are bound to the tetramers may be sorted byfluorescence-controlled cell sorting to isolate reactive cytotoxic Tlymphocytes. The isolated cytotoxic T lymphocytes may then be propagatedin vitro.

In a therapeutic method referred to as adoptive transfer (Greenberg, J.Immunol. 136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynchet al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell59:603-614, 1989), cells presenting the desired complex (e.g. dendriticcells) are combined with cytotoxic T lymphocytes of the patient to betreated, resulting in a propagation of specific cytotoxic T lymphocytes.The propagated cytotoxic T lymphocytes are then administered to apatient having a cellular anomaly characterized by particular abnormalcells presenting the specific complex. The cytotoxic T lymphocytes thenlyse the abnormal cells, thereby achieving a desired therapeutic effect.

Often, of the T cell repertoire of a patient, only T cells with lowaffinity for a specific complex of this kind can be propagated, sincethose with high affinity have been extinguished due to development oftolerance. An alternative here may be a transfer of the T cell receptoritself. For this too, cells presenting the desired complex (e.g.dendritic cells) are combined with cytotoxic T lymphocytes of healthyindividuals or another species (e.g. mouse). This results in propagationof specific cytotoxic T lymphocytes with high affinity if the Tlymphocytes are derived from a donor organism which had no previouscontact with the specific complex. The high affinity T cell receptor ofthese propagated specific T lymphocytes is cloned. If the high affinityT cell receptors have been cloned from another species they can behumanized to a different extent. Such T cell receptors are thentransduced via gene transfer, for example using retroviral vectors, intoT cells of patients, as desired. Adoptive transfer is then carried outusing these genetically altered T lymphocytes (Stanislawski et al., NatImmunol. 2:962-70, 2001; Kessels et al., Nat Immunol. 2:957-61, 2001).

The therapeutic aspects above start out from the fact that at least someof the abnormal cells of the patient present a complex of atumor-associated antigen and an HLA molecule. Such cells may beidentified in a manner known per se. As soon as cells presenting thecomplex have been identified, they may be combined with a sample fromthe patient, which contains cytotoxic T lymphocytes. If the cytotoxic Tlymphocytes lyse the cells presenting the complex, it can be assumedthat a tumor-associated antigen is presented.

Adoptive transfer is not the only form of therapy which can be appliedaccording to the present technology. Cytotoxic T lymphocytes may also begenerated in vivo in a manner known per se. One method usesnonproliferative cells expressing the complex. The cells used here willbe those which usually express the complex, such as irradiated tumorcells or cells transfected with one or both genes necessary forpresentation of the complex (i.e. the antigenic peptide and thepresenting HLA molecule). Various cell types may be used. Furthermore,it is possible to use vectors which carry one or both of the genes ofinterest. Particular preference is given to viral or bacterial vectors.For example, nucleic acids coding for a tumor-associated antigen or fora part thereof may be functionally linked to promoter and enhancersequences which control expression of said tumor-associated antigen or afragment thereof in particular tissues or cell types. The nucleic acidmay be incorporated into an expression vector. Expression vectors may benonmodified extrachromosomal nucleic acids, plasmids or viral genomesinto which exogenous nucleic acids may be inserted. Nucleic acids codingfor a tumor-associated antigen may also be inserted into a retroviralgenome, thereby enabling the nucleic acid to be integrated into thegenome of the target tissue or target cell. In these systems, amicroorganism such as vaccinia virus, pox virus, Herpes simplex virus,retrovirus or adenovirus carries the gene of interest and de facto“infects” host cells. Another preferred form is the introduction of thetumor-associated antigen in the form of recombinant RNA which may beintroduced into cells by liposomal transfer or by electroporation, forexample. The resulting cells present the complex of interest and arerecognized by autologous cytotoxic T lymphocytes which then propagate.

A similar effect can be achieved by combining the tumor-associatedantigen or a fragment thereof with an adjuvant in order to makeincorporation into antigen-presenting cells in vivo possible. Thetumor-associated antigen or a fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The tumor-associatedantigen is processed to produce a peptide partner for the HLA molecule,while a fragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toHLA molecules. Preference is given to administration forms in which thecomplete antigen is processed in vivo by a dendritic cell, since thismay also produce T helper cell responses which are needed for aneffective immune response (Ossendorp et al., Immunol Lett. 74:75-9,2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In general, itis possible to administer an effective amount of the tumor-associatedantigen to a patient by intradermal injection, for example. However,injection may also be carried out intranodally into a lymph node (Maloyet al., Proc Natl Acad Sci USA 98:3299-303, 2001). It may also becarried out in combination with reagents which facilitate uptake intodendritic cells. Preferred tumor-associated antigens comprise thosewhich react with allogenic cancer antisera or with T cells of manycancer patients. Of particular interest, however, are those againstwhich no spontaneous immune responses pre-exist. Evidently, it ispossible to induce against these immune responses which can lyse tumors(Keogh et al., J. Immunol. 167:787-96, 2001; Appella et al., Biomed PeptProteins Nucleic Acids 1:177-84, 1995; Wentworth et al., Mol Immunol.32:603-12, 1995).

The pharmaceutical compositions described according to the presenttechnology may also be used as vaccines for immunization. According tothe present technology, the terms “immunization” or “vaccination” meanan increase in or activation of an immune response to an antigen. It ispossible to use animal models for testing an immunizing effect on cancerby using a tumor-associated antigen or a nucleic acid coding therefor.For example, human cancer cells may be introduced into a mouse togenerate a tumor, and one or more nucleic acids coding fortumor-associated antigens may be administered. The effect on the cancercells (for example reduction in tumor size) may be measured as a measurefor the effectiveness of an immunization by the nucleic acid.

As part of the composition for an immunization, one or moretumor-associated antigens or stimulating fragments thereof areadministered together with one or more adjuvants for inducing an immuneresponse or for increasing an immune response. An adjuvant is asubstance which is incorporated into the antigen or administeredtogether with the latter and which enhances the immune response.Adjuvants may enhance the immune response by providing an antigenreservoir (extracellularly or in macrophages), activating macrophagesand/or stimulating particular lymphocytes. Adjuvants are known andcomprise in a nonlimiting way monophosphoryl lipid A (MPL, SmithKlineBeecham), saponins such as QS21 (SmithKline Beecham), DQS21 SmithKlineBeecham; WO 96/33739), QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells7:178-186, 1997), incomplete Freund's adjuvant, complete Freund'sadjuvant, vitamin E, montanide, alum, CpG oligonucleotides (cf. Kreig etal., Nature 374:546-9, 1995) and various water-in-oil emulsions preparedfrom biologically degradable oils such as squalene and/or tocopherol.Preferably, the peptides are administered in a mixture with DQS21/MPL.The ratio of DQS21 to MPL is typically about 1:10 to 10:1, preferablyabout 1:5 to 5:1 and in particular about 1:1. For administration tohumans, a vaccine formulation typically contains DQS21 and MPL in arange from about 1 μg to about 100 μg.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, interleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds comprisecostimulating molecules provided in the form of proteins or nucleicacids. Examples of such costimulating molecules are B7-1 and B7-2 (CD80and CD86, respectively) which are expressed on dendritic cells (DC) andinteract with the CD28 molecule expressed on the T cells. Thisinteraction provides a costimulation (signal 2) for anantigen/MHC/TCR-stimulated (signal 1) T cell, thereby enhancingpropagation of said T cell and the effector function. B7 also interactswith CTLA4 (CD152) on T cells, and studies involving CTLA4 and B7ligands demonstrate that B7-CTLA4 interaction can enhance antitumorimmunity and CTL propagation (Zheng, P. et al., Proc. Natl. Acad. Sci.USA 95(11):6284-6289 (1998)).

B7 is typically not expressed on tumor cells so that these are noteffective antigen-presenting cells (APCs) for T cells. Induction of B7expression would enable tumor cells to stimulate more effectivelypropagation of cytotoxic T lymphocytes and an effector function.Costimulation by a combination of B7/IL-6/IL-12 revealed induction ofIFN-gamma and Th1-cytokine profile in a T cell population, resulting infurther enhanced T cell activity (Gajewski et al., J. Immunol.154:5637-5648 (1995)).

A complete activation of cytotoxic T lymphocytes and a complete effectorfunction require an involvement of T helper cells via interactionbetween the CD40 ligand on said T helper cells and the CD40 moleculeexpressed by dendritic cells (Ridge et al., Nature 393:474 (1998),Bennett et al., Nature 393:478. (1998), Schonberger et al., Nature393:480 (1998)). The mechanism of this costimulating signal probablyrelates to the increase in B7 production and associated IL-6/IL-12production by said dendritic cells (antigen-presenting Cells).CD40-CD40L interaction thus complements the interaction of signal 1(antigen/MHC-TCR) and signal 2 (B7-CD28).

The use of anti-CD40 antibodies for stimulating dendritic cells would beexpected to directly enhance a response to tumor antigens which areusually outside the range of an inflammatory response or which arepresented by nonprofessional antigen-presenting cells (tumor cells). Inthese situations, T helper and B7-costimulating signals are notprovided. This mechanism could be used in connection with therapiesbased on antigen-pulsed dendritic cells.

The present technology also provides for administration of nucleicacids, polypeptides or peptides. Polypeptides and peptides may beadministered in a manner known per se. In one embodiment, nucleic acidsare administered by ex vivo methods, i.e. by removing cells from apatient, genetic modification of said cells in order to incorporate atumor-associated antigen and reintroduction of the altered cells intothe patient. This generally comprises introducing a functional copy of agene into the cells of a patient in vitro and reintroducing thegenetically altered cells into the patient. The functional copy of thegene is under the functional control of regulatory elements which allowthe gene to be expressed in the genetically altered cells. Transfectionand transduction methods are known to the skilled worker. The presenttechnology also provides for administering nucleic acids in vivo byusing vectors such as viruses and target-controlled liposomes.

In a preferred embodiment, a viral vector for administering a nucleicacid coding for a tumor-associated antigen is selected from the groupconsisting of adenoviruses, adeno-associated viruses, pox viruses,including vaccinia virus and attenuated pox viruses, Semliki Forestvirus, retroviruses, Sindbis virus and Ty virus-like particles.Particular preference is given to adenoviruses and retroviruses. Theretroviruses are typically replication-deficient (i.e. they areincapable of generating infectious particles).

Various methods may be used in order to introduce according to thepresent technology nucleic acids into cells in vitro or in vivo. Methodsof this kind comprise transfection of nucleic acid CaPO₄ precipitates,transfection of nucleic acids associated with DEAE, transfection orinfection with the above viruses carrying the nucleic acids of interest,liposome-mediated transfection, and the like. In particular embodiments,preference is given to directing the nucleic acid to particular cells.In such embodiments, a carrier used for administering a nucleic acid toa cell (e.g. a retrovirus or a liposome) may have a bound target controlmolecule. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell may be incorporated into or attached to the nucleicacid carrier. Preferred antibodies comprise antibodies which bindselectively a tumor-associated antigen. If administration of a nucleicacid via liposomes is desired, proteins binding to a surface membraneprotein associated with endocytosis may be incorporated into theliposome formulation in order to make target control and/or uptakepossible. Such proteins comprise capsid proteins or fragments thereofwhich are specific for a particular cell type, antibodies to proteinswhich are internalized, proteins addressing an intracellular site, andthe like.

The therapeutic compositions of the present technology may beadministered in pharmaceutically compatible preparations. Suchpreparations may usually contain pharmaceutically compatibleconcentrations of salts, buffer substances, preservatives, carriers,supplementing immunity-enhancing substances such as adjuvants, CpG andcytokines and, where appropriate, other therapeutically activecompounds.

The therapeutically active compounds of the present technology may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitonealy, intramuscularly, subcutaneously ortransdermally. Preferably, antibodies are therapeutically administeredby way of a lung aerosol. Antisense nucleic acids are preferablyadministered by slow intravenous administration.

The compositions of the present technology are administered in effectiveamounts. An “effective amount” refers to the amount which achieves adesired reaction or a desired effect alone or together with furtherdoses. In the case of treatment of a particular disease or of aparticular condition characterized by expression of one or moretumor-associated antigens, the desired reaction relates to inhibition ofthe course of the disease. This comprises slowing down the progress ofthe disease and, in particular, interrupting the progress of thedisease. The desired reaction in a treatment of a disease or of acondition may also be delay of the onset or a prevention of the onset ofsaid disease or said condition.

An effective amount of a composition of the present technology willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors.

The pharmaceutical compositions of the present technology are preferablysterile and contain an effective amount of the therapeutically activesubstance to generate the desired reaction or the desired effect.

The doses administered of the compositions of the present technology maydepend on various parameters such as the type of administration, thecondition of the patient, the desired period of administration, etc. Inthe case that a reaction in a patient is insufficient with an initialdose, higher doses (or effectively higher doses achieved by a different,more localized route of administration) may be used.

Generally, doses of the tumor-associated antigen of from 1 ng to 1 mg,preferably from 10 ng to 100 μg are formulated and administered for atreatment or for generating or increasing an immune response. If theadministration of nucleic acids (DNA and RNA) coding fortumor-associated antigens is desired, doses of from 1 ng to 0.1 mg areformulated and administered.

The pharmaceutical compositions of the present technology are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible compositions. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the present technology.Pharmacologically and pharmaceutically compatible salts of this kindcomprise in a nonlimiting way those prepared from the following acids:hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,salicylic, citric, formic, malonic, succinic acids, and the like.Pharmaceutically compatible salts may also be prepared as alkali metalsalts or alkaline earth metal salts, such as sodium salts, potassiumsalts or calcium salts.

A pharmaceutical composition of the present technology may comprise apharmaceutically compatible carrier. According to the presenttechnology, the term “pharmaceutically compatible carrier” refers to oneor more compatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to humans. The term“carrier” refers to an organic or inorganic component, of a natural orsynthetic nature, in which the active component is combined in order tofacilitate application. The components of the pharmaceutical compositionof the present technology are usually such that no interaction occurswhich substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions of the present technology may containsuitable buffer substances such as acetic acid in a salt, citric acid ina salt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the present technology may be in the form of capsules,tablets, lozenges, suspensions, syrups, elixir or in the form of anemulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present technology is described in detail by the figures andexamples below, which are used only for illustration purposes and arenot meant to be limiting. Owing to the description and the examples,further embodiments which are likewise included in the presenttechnology are accessible to the skilled worker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. GPR35 mRNA expression in colon carcinoma biopsies

RT-PCR investigations with DNA-free RNA show GER35 expression in most ofthe colon carcinoma biopsies. By contrast, there is no detectableexpression in normal tissues. (1—Breast, 2—lung, 3—lymph nodes,4—thymus, 5—colon, 6-15 colon carcinoma, 16—neg. control).

FIG. 2. Quantitative PCR analysis of GUCY2C mRNA expression in normaland tumor tissues

Real-time PCR investigation with GUCY2C-specific primers (SEQ ID NO:22-23) shows selective mRNA expression in normal ileum, colon, and inall colon carcinoma biopsies. Distinct quantities of GUCY2C transcriptswere also detected in a colon carcinoma metastasis in the liver.

FIG. 3. Identification of tumor-specific GUCY2C splice variants

PCR products from normal colon tissues and colon carcinomas were cloned,and clones from both groups were checked by restriction analysis (EcoRI) and sequenced.

FIG. 4. Selective SCGB3A expression in normal lung and lung carcinoma

RT-PCR analysis with gene-specific SCGB3A2 primers (SEQ ID NO: 37, 38)shows cDNA amplification exclusively in normal lung (lane 8, 14-15) andin lung carcinoma biopsies (lane 16-24). (1—Liver-N, 2—PBMC-N, 3—lymphnode-N, 4—stomach-N, 5—testis-N, 6—breast-N, 7—kidney-N, 8—lung-N,9—thymus-N, 10—ovary-N, 11—adrenal-N, 12—spleen-N, 14-15—lung-N,16-24—lung carcinoma, 25—negative control).

FIG. 5. Claudin-18A2.1 expression in stomach, esophagus, stomachcarcinoma and pancreatic carcinoma

RT-PCR analysis with claudin-18A2.1-specific primers (SEQ ID NO: 39, 40)showed according to the present technology pronounced claudin-18A2.1expression in 8/10 stomach carcinoma biopsies and in 3/6 pancreaticcarcinoma biopsies. Distinct expression was also detected in stomach andnormal esophageal tissue. In contrast thereto, no expression wasdetected in the ovary and in ovarian carcinoma.

FIG. 6. SLC13A1 expression in the kidney and renal cell carcinoma

RT-PCR analysis with SLC13A1-specific primers (SEQ ID NO: 49, 50) showedexpression in 7/8 renal cell carcinoma samples. Otherwise, transcriptswithin normal tissues were detected exclusively in the kidney.(1-2—kidney, 3-10—renal cell carcinoma, 11—breast, 12—lung, 13—liver,14—colon, 15—lymph nodes, 16—spleen, 17—esophagus, 18—thymus,19—thyroid, 20—PBMCs, 21—ovary, 22—testis).

FIG. 7. CLCA1 expression in colon, colon carcinoma and stomach carcinoma

RT-PCR investigations with CLCA1-specific primers. (SEQ ID NO: 67, 68)confirmed selective expression in the colon and showed high expressionin (3/7) investigated colon carcinoma and (1/3) investigated stomachcarcinoma samples. The other normal tissues (NT) showed no or only veryweak expression.

FIG. 8. FLJ21477 expression in the colon and colon carcinoma

RT-PCR investigations with FLJ21477-specific primers (SEQ ID NO: 69, 70)showed selective expression in the colon and additionally various levelsof expression in (7/12) investigated colon carcinoma samples. The othernormal tissues (NT) showed no expression.

FIG. 9. FLJ20694 expression in the colon and colon carcinoma

RT-PCR investigations with FLJ20694-specific primers (SEQ ID NO: 71, 72)showed selective expression in the colon and additionally various levelsof expression in (5/9) investigated colon carcinoma samples. The othernormal tissues (NT) showed no expression.

FIG. 10. von Ebner expression in stomach, lung and lung carcinoma

RT-PCR investigations with von Ebner-specific primers (SEQ ID NO: 73,74) showed selective expression in the stomach, in the lung and in(5/10) investigated lung carcinoma samples. The other normal tissues(NT) showed no expression.

FIG. 11. Plunc expression in thymus, lung and lung carcinoma

RT-PCR investigations with Plunc-specific primers (SEQ ID NO: 75, 76)showed selective expression in the thymus, in the lung and in (6/10)investigated lung carcinoma samples. The other normal tissues showed noexpression.

FIG. 12. SLC26A9 expression in lung, lung carcinoma and thyroid

RT-PCR investigations with SLC26A9-specific primers (SEQ ID NO: 77, 78)showed selective expression in the lung and in all (13/13) investigatedlung carcinoma samples. The other normal tissues (NT) showed noexpression with the exception of the thyroid.

FIG. 13. THC1005163 expression in stomach, ovary, lung and lungcarcinoma

RT-PCR investigations with a THC1005163-specific primer (SEQ ID NO: 79)and a nonspecific oligo dT tag primer showed expression in stomach,ovary, lung and in (5/9) lung carcinoma biopsies. The other normaltissues (NT) showed no expression.

FIG. 14. LOC134288 expression in kidney and renal cell carcinoma

RT-PCR investigations with LOC134288-specific primers (SEQ ID NO: 80,81) showed selective expression in the kidney and in (5/8) investigatedrenal cell carcinoma biopsies.

FIG. 15. THC943866 expression in kidney and renal cell carcinoma

RT-PCR investigations with THC943866-specific primers (SEQ ID NO: 82,83) showed selective expression in the kidney and in (4/8) investigatedrenal cell carcinoma biopsies.

FIG. 16. FLJ21458 expression in colon and colon carcinoma

RT-PCR investigations with FLJ21458-specific primers (SEQ ID NO: 86, 87)showed selective expression in the colon and in (7/10) investigatedcolon carcinoma biopsies. (1-2—colon, 3—liver, 4—PBMCs, 5—spleen,6—prostate, 7—kidney, 8—ovary, 9—skin, 10—ileum, 11—lung, 12—testis,13-22 colon carcinoma, 23—neg. control).

FIG. 17. Cellular localization of GPR35

Immunofluorescence for detecting the cellular localization of GPR35after transfection of a plasmid that expresses a GPR35-GFP fusionprotein. The arrows identify the membrane-associated fluorescence of thefluorescent GFP.

FIG. 18. Quantitative expression of GPR35

A. Quantitative RT-PCR with GPR35-specific primers (SEQ ID NO: 88, 89)show selective expression in the intestine, in colon tumor samples andin metastases from intestinal tumors. The following normal tissues wereanalyzed: liver, lung, lymph nodes, stomach, spleen, adrenal, kidney,esophagus, ovary, testis, thymus, skin, breast, pancreas, lymphocytes,activated lymphocytes, prostate, thyroid, fallopian tube, endometrium,cerebellum, brain.

B. Prevalence of GPR35 in colon tumors and metastases thereof. GPR35 isexpressed both in the tumor and in metastases in more than 90% of thecases.

FIG. 19. Quantitative expression of GUCY2C

Quantitative RT-PCR with GUCY2C-specific primers (SEQ ID NO: 98, 99)show high and selective expression in normal colonic and gastric tissue(A) and GUCY2C-specific expression in colonic and gastric tumor samples(B). GUCY2C is detectable in 11/12 colon carcinomas and in 7/10 stomachcarcinomas.

FIG. 20. Quantitative expression of SCGB3A2

Quantitative RT-PCR with SCGB3A2-specific primers (SEQ ID NO: 103, 104)show selective expression in lung samples and lung tumor samples. 19/20lung tumor samples are SCGB3A2-positive, and SCGB3A2 is overexpressed bya factor of at least 10 in more than 50% of the samples. The followingnormal tissues were analyzed: liver, lung, lymph nodes, stomach, spleen,adrenal, kidney, esophagus, ovary, testis, thymus, skin, breast,pancreas, lymphocytes, activated lymphocytes, prostate, thyroid,fallopian tube, endometrium, cerebellum, brain.

FIG. 21. Immunofluorescence with SCGB3A2-specific antibodies

COS7 cells were transfected with a plasmid which codes for anSCGB3A2-GFP fusion protein. A. Detection of the transfected fusionprotein with an SCGB3A2-specific rabbit antiserum (immunization with SEQID NO: 105). B. Detection of the transfected fusion protein by GFPfluorescence. C. Superimposition of the two fluorescences from A and B.The yellow color is produced at the points where the two fluorescencesare superimposed and thus demonstrates the specificity of the SCGB3A2antiserum.

FIG. 22. Diagrammatic depiction of claudin-18 splice variants

The two claudin-18 splice variants A1 and A2 differ in the N terminusand show different potential glycosylation sites.

FIG. 23. Quantitative expression of claudin-18, variant A1

Claudin-Al is highly activated in a large number of tumor tissues.Particularly strong expression is found in gastric tumors, lung tumors,pancreatic carcinomas and esophageal carcinomas.

FIG. 24. Quantitative expression of claudin-18, variant A2

Variant A2 is, like variant A1, activated in many tumors.

FIG. 25. Use of claudin-18A2-specific antibodies (extracellular domain)

(Top) Staining of claudin-18A2-positive gastric carcinoma cells (SNU-16)with an antibody which was produced by immunization with a peptide (SEQID NO: 17). Membrane staining appears particularly strongly in thecell/cell interaction regions. A-preimmune, MeOH; B-immune serum MeOH, 5μg/ml;

(Below) Demonstration of the specificity of the antibody bycolocalization analysis in claudin-18A2 GFP-transfected 293T cells.A-Claudin-18A2 GFP; B-anti-claudin-A2; C-superimposition.

FIG. 26. Use of claudin-18A2-specific antibodies (extracellular domain)

Membrane staining of claudin-18A2-positive gastric carcinoma cells(SNU-16) with an antibody which was produced by immunization with apeptide (SEQ ID NO: 113, N-terminally located extracellular domain). Amonoclonal antibody which is directed against E-cadherin was used forcounterstaining. A-antibody; B-counterstaining; C-superimposition.

FIG. 27. Use of antibodies against the C-terminal extracellular domainof claudin-18

(Left, top and below) Membrane staining of claudin-18A2-positive gastriccarcinoma cells (SNU-16) with an antibody which was produced byimmunization with a peptide (SEQ ID NO: 116, C-terminally locatedextra-cellular domain). A monoclonal antibody which is directed againstE-cadherin was used for counter-staining (right top, below).

FIG. 28. Use of claudin-18A1-specific antibodies

(Top) Weak to absent staining of gastric carcinoma cells (SNU-16;claudin18A2 positive) with an antibody which was produced byimmunization with a claudin-18A1-specific peptide (SEQ ID NO: 115).A-anti-E-cadherin; B-anti-claudin-18A1; C-superimposition.

(Below) Demonstration of the specificity of the antibody bycolocalization analysis in claudin-18A1-GFP-transfected 293T cells.A-GFP-claudin-18A1; B-anti-claudin-18A1; C-superimposition.

FIG. 29. Detection of claudin-18A2 in a Western blot.

Western blotting with lysates from various healthy tissues with aclaudin-18A2-specific antibody directed against the epitope with SEQ IDNO: 17. 1—Stomach; 2—testis; 3—skin; 4—breast; 5—liver; 6—colon; 7—lung;8—kidney; 9—lymph nodes.

FIG. 30. Claudin-18A2 Western blotting with samples from stomach andstomach tumors

Lysates from stomach and stomach tumors were blotted and tested using aclaudin-18A2-specific antibody against the epitope having SEQ ID NO: 17.Stomach tumors show a less glycosylated form of claudin-18A2. PNGase Ftreatment of stomach lysates leads to the formation of thelow-glycosylated form.

Left: 1—stomach No #A; 2—stomach Tu #A; 3—stomach No #B; 4—stomach Tu #B

Right: 1—stomach No #A; 2—stomach No #B; 3—stomach No #B+PNGase F;4—stomach Tu #C; 5—stomach Tu #D; 6—stomach Tu #D+PNGase F

FIG. 31. Expression of claudin-18 in lung tumors

Low-glycosylated claudin-18A2 variants were detected in lung tumors inaccordance with FIG. 30. 1—Stomach No; 2—stomach Tu; 3-9—lung Tu.

FIG. 32. Immunohistochemlcal analysis of claudin-18 usingclaudin-18A2-specific antibodies in stomach tumor tissue

FIG. 33. Indirect immunofluorescence of stomach-specific Snu16 cellswith a claudin-18-specific polyclonal antiserum

A. Staining with a preimmune serum generated before the immunization; B.Staining with the claudin-18-specific serum.

FIG. 34. Quantitative expression of SLC13A1

Quantitative RT-PCR with SLC13A1-specific primers (SEQ ID NO: 121, 122)show high and selective expression in normal kidney tissue (A) andSLC13A1-specific expression in renal cell carcinomas (B). SLC13A1transcription is detectable in 5/8 renal cell carcinomas.

FIG. 35. Cellular localization of SLC13A1

Immunofluorescence to demonstrate the cellular localization of SLC13A1after transfection of a plasmid which provides an SLC13A1-GFP fusionprotein. The membrane-associated fluorescence of the SLC13A1 fusionprotein is to be seen clearly (as ring around the transfected cell).

FIG. 36. Quantitative expression of CLCA1

Quantitative RT-PCR with CLCA1-specific primers (SEQ ID NO: 125, 126)show high and selective expression in normal colonic tissue and stomachtissue (A) and CLCA1-specific expression in colonic and gastric tumorsamples (8). CLCA1 is detectable in 6/12 colon carcinomas and in 7/10stomach carcinomas.

FIG. 37. Quantitative expression of FLJ21477

Quantitative RT-PCR with FLJ21477-specific primers (SEQ ID NO: 127, 128)show high and selective expression in normal colonic and gastric tissueand weak expression in thymus, esophagus and brain (A) and theFLJ21477-specific expression in colonic tumor samples (B). FLJ21477 isdetectable in 11/12 colon carcinomas.

FIG. 38. Quantitative expression of FLJ20694

Quantitative RT-PCR with FLJ20694-specific primers (SEQ ID NO: 129, 130)show high and selective expression in normal colonic and gastric tissue(A) and FLJ20694-specific overexpression in colonic and gastric tumorsamples (B). FLJ20694 is detectable in 11/12 colon carcinomas and in7/10 stomach carcinomas.

FIG. 39. Quantitative expression of FLJ21458

Quantitative RT-PCR with FLJ21458-specific primers (SEQ ID NO: 133, 134)show selective expression in testis, gastric and intestinal tissue. Inaddition, FLJ21458-specific transcripts were detectable in 20/20 colonictumors and in 7/11 colonic metastases. The following normal tissues wereanalyzed: liver, lung, lymph nodes, spleen, adrenal, kidney, esophagus,ovary, testis, thymus, skin, breast, pancreas, lymphocytes, activatedlymphocytes, prostate, thyroid, fallopian tube, endometrium, cerebellum,brain.

FIG. 40. Immunofluorescence with FLJ21458-specific antibodies

(Top) 293 cells were transfected with a plasmid which codes for anFLJ21458-GFP fusion protein. A: detection of the transfected fusionprotein with an FLJ21458-specific rabbit antiserum (immunization withSEQ ID NO: 136). B: detection of the transfected fusion protein by GFPfluorescence. C: superimposition of the two fluorescences from A and B.The yellow color is produced at the points where the two fluorescencesare superimposed and thus demonstrates the specificity of the FLJ21458antiserum.

(Below) Analysis of Snu16 cells which endogenously synthesize FLJ21458.A: protein detection using an FLJ21458-specific rabbit antiserum(immunization with SEQ ID NO: 136). B: detection of the membrane proteinE-cadherin. C: superimposition of the two fluorescences from A and B.The yellow color is produced at the points where the two fluorescencesare superimposed, and demonstrates the membrane localization ofFLJ21458.

FIG. 41A-41Q. Sequences

The sequences to which reference is made herein are shown.

EXAMPLES

Material and Methods

The terms “in silico”, “electronic” and “virtual cloning” refer solelyto the utilization of methods based on databases, which may also be usedto simulate laboratory experimental processes.

Unless expressly defined otherwise, all other terms and expressions areused so as to be understood by the skilled worker. The techniques andmethods mentioned are carried out in a manner known per se and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Edition (1989) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. All methods including the use of kitsand reagents are carried out according to the manufacturers'information.

Datamining-Based Strategy for Determining New Tumor-Associated Genes

Two in silico strategies, namely GenBank keyword search and thecDNAxProfiler, were combined. Utilizing the NCBI ENTREZ Search andRetrieval System (http://www.ncbi.nlm.nih.gov/Entrez), a GenBank searchwas carried out for candidate genes annotated as being specificallyexpressed in specific tissues (Wheeler et al., Nucleic Acids Research28:10-14, 2000).

Carrying out queries with keywords such as “colon-specific gene”,“stomach-specific gene” or “kidney-specific gene”, candidate genes (GOI,genes of interest) were extracted from the databases. The search wasrestricted to part of the total information of these databases by usingthe limits “homo sapiens”, for the organism, and “mRNA”, for the type ofmolecule.

The list of the GOI found was curated by determining different names forthe same sequence and eliminating such redundancies.

All candidate genes obtained by the keyword search were in turn studiedwith respect to their tissue distribution by the “electronic Northern”(eNorthen) method. The eNorthern is based on aligning the sequence of aGOI with an EST (expressed sequence tag) database (Adams et al., Science252:1651, 1991) (http://www.ncbi.nlm.nih.gov/BLAST). The tissue originof each EST which is found to be homologous to the inserted GOI can bedetermined and in this way the sum of all ESTs produces a preliminaryassessment of the tissue distribution of the GOI. Further studies werecarried out only with those GOI which had no homologies to EST from nonorgan-specific normal tissues. This evaluation also took into accountthat the public domain contains wrongly annotated cDNA libraries(Scheurle et al., Cancer Res. 60:4037-4043, 2000)(www.fau.edu/cmbb/publications/cancergenes6.htm).

The second datamining method utilized was the cDNA xProfiler of the NCBICancer Genome Anatomy Project(http://cgap.nci.nih.gov/Tissues/xProfiler) (Hillier et al., GenomeResearch 6:807-828, 1996; Pennisi, Science 276:1023-1024, 1997). Thisallows pools of transcriptomes deposited in databases to be related toone another by logical operators. We have defined a pool A to which allexpression libraries prepared for example from colon were assigned,excluding mixed libraries. All cDNA libraries prepared from normaltissues other than colon were assigned to pool B. Generally, all cDNAlibraries were utilized independently of underlying preparation methods,but only those with a size >1000 were admitted. Pool B was digitallysubtracted from pool A by means of the BUT NOT operator. The set of GOIfound in this manner was also subjected to eNorthern studies andvalidated by a literature research.

This combined datamining includes all of the about 13 000 full-lengthgenes in the public domain and predicts out of these genes havingpotential organ-specific expression.

All other genes were first evaluated in normal tissues by means ofspecific RT-PCR. All GOI which had proved to be expressed in non-organspecific normal tissues had to be regarded as false-positives and wereexcluded from further studies. The remaining ones were studied in alarge panel of a wide variety of tumor tissues. The antigens depictedbelow proved here to be activated in tumor cells.

RNA Extraction, Preparation of Poly-d(T) Primed cDNA and ConventionalRT-PCR Analysis

Total RNA was extracted from native tissue material by using guanidiumisothiocyanate as chaotropic agent (Chomczynski Sacchi, Anal. Biochem.162:156-9, 1987). After extraction with acidic phenol and precipitationwith isopropanol, said RNA was dissolved in DEPC-treated water.

First strand cDNA synthesis from 2-4 μg of total RNA was carried out ina 20 μl reaction mixture by means of Superscript II (Invitrogen),according to the manufacturer's information. The primer used was adT(18) oligonucleotide. Integrity and quality of the cDNA were checkedby amplification of p53 in a 30 cycle PCR (sense CGTGAGCGCTTCGAGATGTTCCG(SEQ ID NO. 138), antisense CCTAACCAGCTGCCCAACTGTAG (SEQ ID NO. 139),hybridization temperature 67° C.

An archive of first strand cDNA was prepared from a number of normaltissues and tumor entities. For expression studies, 0.5 μl of thesecDNAs was amplified in a 30 μl reaction mixture, using GOI-specificprimers (see below) and 1 U of HotStarTaq DNA polymerase (Qiagen). Eachreaction mixture contained 0.3 mM dNTPs, 0.3 μM of each primer and 3 μlof 10× reaction buffer. The primers were selected so as to be located intwo different exons, and elimination of the interference bycontaminating genomic DNA as the reason for false-positive results wasconfirmed by testing nonreverse-transcribed DNA as template. After 15minutes at 95° C. to activate the HotStarTaq DNA polymerase, 35 cyclesof PCR were carried out (1 min at 94° C., 1 min at the particularhybridization temperature, 2 min at 72° C. and final elongation at 72°C. for 6 min). 20 μl of this reaction were fractionated and analyzed onan ethidium bromide-stained agarose gel.

The following primers were used for expression analysis of thecorresponding antigens at the hybridization temperature indicated.

GPR35 (65° C.) Sense:  (SEQ ID NO. 20) 5′-AGGTACATGAGCATCAGCCTG-3′Antisense:  (SEQ ID NO. 21) 5′-GCAGCAGTTGGCATCTGAGAG-3′ GUCY2C (62° C.)Sense:  (SEQ ID NO. 22) 5′-GCAATAGACATTGCCAAGATG-3′ Antisense: (SEQ ID NO. 23) 5′-AACGCTGTTGATTCTCCACAG-3′ SCGB3A2 (66° C.) Sense: (SEQ ID NO. 37) 5′-CAGCCTTTGTAGTTACTCTGC-3′ Antisense:  (SEQ ID NO. 38)5′-TGTCACACCAAGTGTGATAGC-3′ Claudin18A2 (68° C.) Sense1: (SEQ ID NO. 39) 5′-GGTTCGTGGTTTCACTGATTGGGATTGC-3′ Antisense1: (SEQ ID NO. 40) 5′-CGGCTTTGTAGTTGGTTTCTTCTGGTG-3′ Sense2: (SEQ ID NO. 107) 5′-TGTTTTCAACTACCAGGGGC-3′ Antisense2: (SEQ ID NO. 108) 5′-TGTTGGCTTTGGCAGAGTCC-3′ Claudin18A1 (64° C.) Sense: (SEQ ID NO. 109) 5′-GAGGCAGAGTTCAGGCTTCACCGA-3′ Antisense: (SEQ ID NO. 110) 5′-TGTTGGCTTTGGCAGAGTCC-3′ SLC13A1- (64° C.) Sense: (SEQ ID NO. 50) 5′-CAGATGGTTGTGAGGAGTCTG-3′ Antisense:  (SEQ ID NO. 49)5′-CCAGCTTTAACCATGTCAATG-3′ CLCA1 (62° C.) Sense:  (SEQ ID NO. 67)5′-ACACGAATGGTAGATACAGTG-3′ Antisense:  (SEQ ID NO. 68)5′-ATACTTGTGAGCTGTTCCATG-3′ FLJ21477 (68° C.) Sense:  (SEQ ID NO. 69)5′-ACTGTTACCTTGCATGGACTG-3′ Antisense:  (SEQ ID NO. 70)5′-CAATGAGAACACATGGACATG-3′ FLJ20694 (64° C.) Sense:  (SEQ ID NO. 140)5′-CCATGAAAGCTCCATGTCTA-3′ Antisense:  (SEQ ID NO. 72)5′-AGAGATGGCACATATTCTGTC-3′ Ebner (70° C.) Sense:  (SEQ ID NO. 73)5′-ATCGGCTGAAGTCAAGCATCG-3′ Antisense:  (SEQ ID NO. 74)5′-TGGTCAGTGAGGACTCAGCTG-3′ Plunc (55° C.) Sense:  (SEQ ID NO. 75)5′-TTTCTCTGCTTGATGCACTTG-3′ Antisense:  (SEQ ID NO. 76)5′-GTGAGCACTGGGAAGCAGCTC-3′ SLC26A9 (67° C.) Sense:  (SEQ ID NO. 141)5′-GGCAAATGCTAGAGACGTGA-3′ Antisense:  (SEQ ID NO. 78)5′-AGGTGTCCTTCAGCTGCCAAG-3′ THC1005163 (60° C.) Sense:  (SEQ ID NO. 79)5′-GTTAAGTGCTCTCTGGATTTG-3′ LOC134288 (64° C.) Sense:  (SEQ ID NO. 80)5′-ATCCTGATTGCTGTGTGCAAG-3′ Antisense:  (SEQ ID NO. 81)5′-CTCTTCTAGCTGGTCAACATC-3′ THC943866 (59° C.) Sense:  (SEQ ID NO. 82)5′-CCAGCAACAACTTACGTGGTC-3′ Antisense:  (SEQ ID NO. 83)5′-CCTTTATTCACCCAATCACTC-3′ FLJ21458 (62° C.) Sense:  (SEQ ID NO. 86)5′-ATTCATGGTTCCAGCAGGGAC-3′ Antisense:  (SEQ ID NO. 87)5′-GGGAGACAAAGTCACGTACTC-3′.

Preparation of Random Hexamer-Primed cDNA and Quantitative Real-Time PCR

The expression of several genes was quantified by real-time PCR. The PCRproducts were detected using SYBR Green as intercalating reporter dye.The reporter fluorescence of SYBR Green is suppressed in solution andthe dye is active only after binding to double-stranded DNA fragments.The increase in the SYBR Green fluorescence as a result of the specificamplification using GOI-specific primers after each PCR cycle isutilized for quantification. Expression of the target gene is quantifiedabsolutely or relative to the expression of a control gene with constantexpression in the tissues to be investigated. Expression was measuredafter standardization of the samples against 18s RNA as so-calledhousekeeping gene using the ΔΔ-C_(t) method (PE Biosystems, USA). Thereactions were carried out in duplicates and determined in triplicates.The QuantiTect SYBR Green PCR kit (Qiagen, Hilden) was used inaccordance with the manufacturer's instructions. The cDNA wassynthesized using the high capacity cDNA Archive Kit (PE Biosystems,USA) with use of hexamer primers in accordance with the manufacturer'sinstructions. Each 5 μl portions of the diluted cDNA were employed in atotal volume of 25 μl for the PCR: sense primer 300 nM, antisense primer300 nM; initial denaturation 95° C. for 15 min; 95° C. for 30 sec;annealing for 30 sec; 72° C. for 30 sec; 40 cycles. The sequences of theprimers used are indicated in the respective examples.

Cloning and Sequence Analysis

Cloning of full-lengths and gene fragments took place by conventionalmethods. To ascertain the sequence, corresponding antigenes wereamplified using the proofreading polymerase pfu (Stratagene). Aftercompletion of the PCR, adenosine was ligated by means of HotStarTaq DNApolymerase to the ends of the amplicon in order to clone the fragmentsin accordance with the manufacturer's instructions into the TOPO-TAvector. The sequencing was carried out by a commercial service. Thesequences were analysed using conventional prediction programs andalgorithms.

Western Blotting

Cells from cell culture (endogenous expression of the target gene orsynthesis of the target protein after transfection of an expressionvector which encodes the target protein) or tissue samples which mightcontain the target protein are lysed in a 1% SDS solution. The SDSdenatures the proteins present in the lysate. The lysates of anexperimental mixture are fractionated according to size byelectrophoresis on 8-15% denaturing polyacrylamide gels (containing 1%SDS) depending on the expected protein size (SDS polyacrylamide gelelectrophoresis, SDS-PAGE). The proteins are then transferred by thesemi-dry electroblotting method (Biorad) to nitrocellulose membrane(Schleicher & Schtill) on which the desired protein can be detected. Forthis purpose, the membrane is initially blocked (e.g. with milk powder)and then incubated with the specific antibody in a dilution of1:20-1:200 (depending on the specificity of the antibody) for 60minutes. After a washing step, the membrane is incubated with a secondantibody coupled to a marker (e.g. enzymes such as peroxidase oralkaline phosphatase) which recognizes the first antibody. After afurther washing step, subsequently the target protein is visualized in acolor or chemiluminescence reaction, on the membrane by means of anenzyme reaction (e.g. ECL, Amersham Bioscience). The result isdocumented by photographing with a suitable camera.

Analysis of protein modifications usually takes place by Westernblotting. Glycosilations, which usually have a size of several kDa, leadto a larger total mass of the target protein, which can be fractionatedin the SDS-PAGE. To detect specific 0- and N-glycosidic linkages,protein lysates from tissues or cells are incubated before denaturationby SDS with 0- or N-glycosidases (in accordance with their respectivemanufacturer's instructions, e.g. PNgase, endoglycosidase F,endoglycosidase H, Roche Diagnostics). This is followed by Westernblotting as described above. Thus, if there is a reduction in the sizeof a target protein after incubation with a glycosidase it is possibleto detect a specific glycosilation and, in this way, also analyse thetumor specificity of a modification. The exact position of theglycosilated amino acid can be predicted with algorithms and predictionprograms.

Immunofluorescence

Cells of established cell lines which either synthesize the targetprotein endogenously (detection of the RNA in RT-PCR or of the proteinby Western blotting) or else have been transfected with plasmid DNAbefore the IF are used. A wide variety of methods (e.g. electroporation,liposome-based transfection, calcium phosphate precipitation) are wellestablished for transfecting cell lines with DNA (e.g. Lemoine et al.Methods Mol. Biol. 1997; 75: 441-7). The transfected plasmid may in theimmunofluorescence encode the unmodified protein or else couple variousamino acid markers to the target protein. The most important markersare, for example, the fluorescing “green fluorescent protein” (GFP) inits various differentially fluorescing forms and short peptide sequencesof 6-12 amino acids for which high-affinity and specific antibodies areavailable. Cells which synthesize the target protein are fixed withparaformaldehyde, saponin or methanol. The cells can then if required bepermeabilized by incubation with detergents (e.g. 0.2% Triton X-100).After the fixation/permeabilization, the cells are incubated with aprimary antibody which is directed against the target protein or againstone of the coupled markers. After a washing step, the mixture isincubated with a second antibody coupled to a fluorescent marker (e.g.fluorescin, Texas Red, Dako) which binds to the first antibody. Thecells labeled in this way are then covered with a layer of glycerol andanalysed with the aid of a fluorescence microscope according to themanufacturer's instructions. Specific fluorescence emissions areachieved in this case by specific excitation depending on the substancesemployed. The analysis normally allows reliable localization of thetarget protein, the antibody quality and the target protein beingconfirmed in double stainings to stain in addition to the target proteinalso the coupled amino acid markers or other marker proteins whoselocalization has been described in the literature. GFP and itsderivatives represents a special case that can be directly excited anditself fluoresces, so that no antibodies are necessary for thedetection.

Immunohistochemistry

IHC serves specifically for (1) being able to estimate the amount oftarget protein in tumor and normal tissues, (2) analysing how many cellsin the tumor and healthy tissue synthesize the target gene, and/or (3)defining the cell type in a tissue (tumor, healthy cells) in which thetarget protein is detectable. Different protocols must be used dependingon the individual antibody (e.g. “Diagnostic Immunohistochemistry byDavid J., MD Dabbs ISBN: 0443065667” or in “Microscopy,Immunohistochemistry, and Antigen Retrieval Methods: For Light andElectron Microscopy ISBN: 0306467704”).

Immunohistochemistry (IHC) on specific tissue samples serves to detectprotein in the corresponding tissue. The aim of this method is toidentify the localization of a protein in a functionally intact tissueaggregate. IHC serves specifically for (1) being able to estimate theamount of target protein in tumor and normal tissues, (2) analysing howmany cells in tumor and healthy tissue synthesize the target gene, and(3) defining the cell type in a tissue (tumor, healthy cells) in whichthe target protein is detectable. Alternatively, the amounts of proteinof a target gene can be quantified by tissue immunofluorescence using adigital camera and suitable software (e.g. Tillvision, Till-photonics,Germany). The technology has frequently been published, and details ofstaining and microscopy can therefore be found for example in“Diagnostic Immunohistochemistry” by David J., MD Dabbs ISBN: 0443065667or “Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: ForLight and Electron Microscopy” ISBN: 0306467704. It should be notedthat, because of the properties of antibodies, different protocols haveto be used (an example is described below) in order to obtain a validresult.

Ordinarily, histologically defined tumor tissues and, as reference,comparable healthy tissues are employed in the IHC. It is moreoverpossible to use as positive and negative controls cell lines in whichthe presence of the target gene is known through RT-PCR analyses. Abackground control must always be included.

Fixed tissue (e.g. fixation with aldehyde-containing substances,formaldehyde, paraformaldehyde or in alcoholic solutions) orshock-frozen tissue pieces with a thickness of 1-m are applied to aglass-support. Paraffin-embedded samples are deparaffinated for examplewith xylene. The samples are washed with TBS-T and blocked in serum.This is followed by incubation with the first antibody (dilution: 1:2 to1:2000) for 1-18 hours, with affinity-purified antibodies normally beingused. A washing step is followed by incubation with a second antibodywhich is coupled to an alkaline phosphatase (alternative: for exampleperoxidase), and. is directed against the first antibody, for about30-60 minutes. This is followed by color reaction using color substrateswhich are converted by the bound enzymes (cf for example, Shi et al., J.Histochem. Cytochem. 39: 741-748, 1991; Shin et al., Lab. Invest. 64:693-702, 1991). To demonstrate the antibody specificity, the reactioncan be blocked by previous addition of the immunogen.

Immunization

(See also Monoclonal Antibodies: A Practical Approach by PhilipShepherd, Christopher Dean isbn 0-19-963722-9; Antibodies: A LaboratoryManual by Ed Harlow, David Lane ISBN: 0879693142; Using Antibodies: ALaboratory Manual: Portable Protocol NO. by Edward Harlow, David Lane,Ed Harlow ISBN: 0879695447). The process for preparing antibodies isdescribed briefly below, and details can be found in the citedpublications. Firstly, animals (e.g. rabbits) are immunized by a firstinjection of the desired target protein. The animal's immune response tothe immunogen can be enhanced by a second or third immunization within adefined period (about 2-4 weeks after the preceding immunization). Againafter various defined periods (first bleeding after 4 weeks, then aboutevery 2 weeks with a total of up to 5 samplings), blood is taken fromthe animals, and an immune serum is obtained therefrom.

The animals are usually immunized by one of four well-establishedmethods, with other methods also being available. It is moreoverpossible to immunize with peptides which are specific for the targetprotein, with the complete protein or with extracellular partialsequences of a protein which can be identified experimentally or viaprediction programs.

-   -   (1) In the first case, peptides (length: 8-12 amino acids)        conjugated to KLH (keyhole limpet hemocyanin) are synthesized by        a standardized in vitro method, and these peptides are used for        the immunization. Usually, 3 immunizations are carried out with        a concentration of 5-1000 μg/immunization. The immunization can        also be carried out as service from service providers.    -   (2) Alternatively, the immunization can be carried out with        recombinant proteins. For this purpose, the cloned DNA of the        target gene is cloned into an expression vector, and the target        protein is synthesized in analogy to the conditions of the        particular manufacturer (e.g. Roche Diagnostics, Invitrogen,        Clontech, Qiagen) for example cell-free in vitro, in bacteria        (e.g. E. coli), in yeast (e.g. S. pombe), in insect cells or in        mammalian cells. After synthesis in one of the systems, the        target protein is purified, the purification in this case        usually taking place by standardized chromatographic methods. It        is also possible in this connection to use for the immunization        proteins which have a molecular anchor as aid for purification        (e.g. His tag, Qiagen; FLAG tag, Roche Diagnostics; Gst fusion        proteins). A large number of protocols is to be found for        example in the “Current Protocols in Molecular Biology”, John        Wiley & Sons Ltd., Wiley Interscience.    -   (3) If a cell line which synthesizes the desired protein        endogenously is available, this cell line can also be used to        produce the specific antiserum. In this case, the immunization        takes place in 1-3 injections in each case with about 1-5×10⁷        cells.    -   (4) The immunization can also take place by injection of DNA        (DNA immunization). For this purpose, the target gene is        initially cloned into an expression vector so that the target        sequence is under the control of a strong eukaryotic promoter        (e.g. CMV promoter). Subsequently, 5-100 μg of DNA are        transferred as immunogen using a “gene gun” into capillary        regions with a strong blood flow in an organism (e.g. mouse,        rabbit). The transferred DNA is taken up by the animal's cells,        the target gene is expressed, and the animal finally develops an        immune response to the target gene (Jung et al., Mol Cells        12:41-49, 2001; Kasinrerk et al., Hybrid Hybridomics 21:287-293,        2002).

Quality Control of the Polyclonal Serum or Antibody

Assays based on cell culture with subsequent Western blotting are mostsuitable for demonstrating specificity (various variations are describedfor example in “Current Protocols in Protein Chemistry”, John Wiley &Sons Ltd., Wiley InterScience). For the demonstration, cells aretransfected with a cDNA, which is under the control of a strongeukaryotic promoter (e.g. cytomegalovirus promoter), for the targetprotein. A wide variety of methods (e.g. electroporation, liposome-basedtransfection, calcium phosphate precipitation) are well established fortransfecting cell lines with DNA (e.g. Lemoine et al., Methods Mol.Biol. 75:441-7, 1997). It is also possible alternatively to use celllines which express the target gene endogenously (demonstration bytarget gene-specific RT-PCR). As control, in the ideal case homologousgenes are also transfected in the experiment, in order to be able todemonstrate in the following Western blot the specificity of theanalysed antibody.

In the subsequent Western blot, cells from cell culture or tissuesamples which might contain the target protein are lysed in a 1% SDSsolution, and the proteins are denatured thereby. The lysates arefractionated according to size by electrophoresis on 8-15% denaturingpolyacrylamide gels (contain 1% SDS) (SDS polyacrylamide gelelectrophoresis, SDS-PAGE). The proteins are then transferred by one ofa plurality of blotting methods (e.g. semi-dry electroblot; Biorad) to aspecific membrane (e.g. nitrocellulose, Schleicher & Schill). Thedesired protein can be visualized on this membrane. For this purpose,the membrane is first incubated with the antibody which recognizes thetarget protein (dilution about 1:20-1:200, depending on the specificityof the antibody) for 60 minutes. After a washing step, the membrane isincubated with a second antibody which is coupled to a marker (e.g.enzymes such as peroxidase or alkaline phosphatase) and which recognizesthe first antibody. It is then possible in a color or chemiluminescentreaction to visualize the target protein on the membrane (e.g. ECL,Amersham Bioscience). An antibody with a high specificity for the targetprotein should in the ideal case recognize only the desired proteinitself.

Various methods are used to confirm the membrane localization of thetarget protein identified in the in silica approach. An important andwell-established method using the antibodies described above isimmuno-fluorescence (IF). Cells of established cell lines which eithersynthesize the target protein (detection of the RNA in an RT-PCR or ofthe protein in a Western blot) or else have been transfected withplasmid DNA are used for this. A wide variety of methods (e.g.electroporation, liposome-based transfection, calcium phosphateprecipitation) are well established for transfection of cell lines withDNA (e.g. Lemoine et al., Methods Mol. Biol. 75:441-7, 1997). Theplasmid transfected into the cells can in the immunofluorescence encodethe unmodified protein or else couple various amino acid markers to thetarget protein. The principal markers are, for example, the fluorescent“green fluorescent protein” (GFP) in its various differentiallyfluorescent forms, short peptide sequences of 6-12 amino acids for whichhigh-affinity and specific antibodies are available, or the short aminoacid sequence Cys-Cys-X-X-Cys-Cys which can bind via its cysteinespecific fluorescent substances (Invitrogen). Cells which synthesize thetarget protein are fixed for example with paraformaldehyde or methanol.The cells can then, if required, be permeabilized by incubation withdetergents (e.g. 0.2% Triton X-100). The cells are then incubated with aprimary antibody which is directed against the target protein or againstone of the coupled markers. After a washing step, the mixture isincubated with a second antibody which is coupled to a fluorescentmarker (e.g. fluorescin, Texas Red, Dako) and which binds to the, firstantibody. The cells labeled in this way are then covered with a layer ofglycerol and analysed with the aid of a fluorescence microscopeaccording to the manufacturer's instructions. Specific fluorescenceemissions are achieved in this case by specific excitation depending onthe substances employed. The analysis usually permits reliablelocalization of the target protein, the antibody quality and the targetprotein being confirmed in double stainings to stain in addition to thetarget protein also the coupled amino acid markers or other markerproteins whose localization has already been described in theliterature. GFP and its derivatives represents a special case, beingexcitable directly and themselves fluorescing. The membranepermeability, which can be controlled through the use of detergents,permits demonstration in the immunofluorescence of whether animmunogenic epitope is located inside or outside the cell. Theprediction of the selected proteins can thus be supportedexperimentally. An alternative possibility is to detect extracellulardomains by means of flow cytometry. For this purpose, cells are fixedunder non-permeabilizing conditions (e.g. with PBS/Na azide/2%. FCS/5 mMEDTA) and analysed in a flow cytometer in accordance with themanufacturer's instructions. Only extracellular epitopes can berecognized by the antibody to be analysed in this method. A differencefrom immunofluorescence is that it is possible to distinguish betweendead and living cells by use of, for example, propidium iodide or Trypanblue, and thus avoid false-positive results.

Affinity Purification

Purification of the polyclonal sera took place in the case of thepeptide antibodies entirely, or in the case of the antibodies againstrecombinant proteins in part, as service by the contracted companies.For this purpose; in both cases, the appropriate peptide or recombinantprotein was covalently bonded to a matrix, and the latter was, after thecoupling, equilibrated with a native buffer (PBS: phosphate bufferedsaline) and then incubated with the crude serum. After a further PBSwashing step, the antibody was eluted with 100 mM glycine, pH 2.7, andthe eluate was immediately neutralized in 2M TRIS, pH 8. The antibodiespurified in this way could then be employed for specific detection ofthe target proteins both by Western blotting and by immunofluorescence.

Preparation of EGFP Transfectants

For the immunofluorescence microscopy of heterologously expressedtumor-associated antigens, the complete ORF of the antigens was clonedin pEGFP-Cl and pEGFP-N3 vectors (Clontech). CHO and NIH3T3 cellscultivated on slides were transfected with the appropriate plasmidconstructs using Fugene transfection reagent (Roche) in accordance withthe manufacturer's instructions and, after 12-24 h, analysed byimmunofluorescence microscopy.

Example 1: Identification of GPR35 as Diagnostic and Therapeutic CancerTarget

GPR35 (SEQ ID NO: 1) and its translation product (SEQ ID NO: 9) havebeen described as putative G protein-coupled receptor. The sequence ispublished in Genbank under accession No. AF089087. This transcript codesfor a protein of 309 amino acids with a molecular weight of 34 kDa. Itwas predicted that GPR35 belongs to the superfamily of G protein-coupledreceptors with 7 transmembrane domains (O'Dowd et al., Genomics47:310-13, 1998). In order to confirm the predicted localization ofGPR35 in the cell, the protein was fused to eGFP as reporter moleculeand, after transfection of the appropriate plasmid, expressedheterologously in 293 cells. The localization was then analysed in afluorescence microscope. It was confirmed according to the presenttechnology that GPR35 is an integral transmembrane molecule (FIG. 17).Investigation to date on human GPR35 (see, inter alia, Horikawa Y, OdaN, Cox N J, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner T H,Mashima H, Schwarz P E, del Bosque-Plata L, Horikawa Y, Oda Y, YoshiuchiI, Colilla S, Polonsky K S, Wei S, Concannon P, Iwasaki N, Schulze J,Baler L J, Bogardus C, Groop L, Boerwinkle E, Hanis C L, Bell G I NatGenet. 2000 October; 26(2): 163-75) suggested that GPR35 is activated inmany healthy tissues. The reading frame of the gene comprises a singleexon. According to the present technology, a gene-specific primer pair(SEQ ID NO: 20, 21). for GPR35 was used in RT-PCR analyses to amplifycDNA in the colon and in colon carcinoma (13/26). By contrast, nosignificant expression is detectable in other normal tissues. Because ofthe particular fact that GPR35 consists of a single exon, genomic DNAimpurities cannot be detected with intron-spanning primers. In order topreclude genomic contamination of the RNA samples, therefore, all RNAswere treated with DNAse. GPR35 transcripts were detected according tothe present technology only in the colon, in the rectum, in the testisand in colon carcinomas using DNA-free RNA

TABLE 1 GPR35 expression in normal tissues Normal tissue ExpressionBrain − Cerebellum − Myocardium − Skeletal muscle − Rectum ++ Stomach −Colon ++ Pancreas − Kidney − Testis − Thymus − Mammary glands − Ovary −Uterus n.d. Skin − Lung − Thyroid − Lymph nodes − Spleen − PBMC −Adrenal − Esophagus − Small intestine + Prostate − (nd = not determined)

The selective and high expression of GPR35 transcripts in normal colonictissue and in colon carcinoma biopsies (FIG. 1) was not previously knownand can be utilized according to the present technology for moleculardiagnostic methods such as RT-PCR for detecting disseminating tumorcells in the serum and bone marrow and for detecting disseminating tumorcells in the serum and bone marrow and for detecting metastases in othertissues. Quantitative RT-PCR with specific primers (SEQ ID NO: 88 and89) also confirms that GPR35 is a highly selective intestine-specificdifferentiation antigen which is also contained in intestinal tumors andin intestinal tumor metastases. In some intestinal tumors, it is in factoverexpressed by one log compared with normal intestine (FIG. 18).Antibodies were produced by immunizing rabbits for detecting GPR35protein. The following peptides were used to propagate these antibodies:

SEQ ID NO: 90  GSSDLTWPPAIKLGC (AA 9-23) SEQ ID NO: 91: DRYVAVRHPLRARGLR (AA 112-127) SEQ ID NO: 92: VAPRAKAHKSQDSLC (C terminus) SEQ ID NO: 93 CFRSTRHNFNSMR (extracell. domain 2)Stainings with these antibodies for example in a Western blot confirmthe expression in tumors. All 4 extracellular domains of GPR35 (positionof the predicted extracellular domains in the sequence of SEQ ID NO: 9AA 1-22 (SEQ ID NO: 94); AA 81-94 (SEQ ID NO: 95); AA 156-176 (SEQ. IDNO: 96); AA 280-309 (SEQ ID NO: 97)) can be used according to thepresent technology as target structures of monoclonal antibodies. Theseantibodies bind specifically to the cell surface of tumor cells and canbe used both for diagnostic and for therapeutic methods. Overexpressionof GPR35 in tumors provides additional support for such a use. Inaddition, the sequences coding for proteins can be used according to thepresent technology as vaccine (RNA, DNA, peptide, protein) for inducingtumor-specific immune responses (T-cell and B-cell-mediated immuneresponses). In addition, it has surprisingly been found that a furtherstart codon exists 5′ in front of the generally known start codon andexpresses an N-terminally extended protein.

It has thus been found according to the present technology that GPR35, aprotein which was previously described as expressed ubiquitously, istumor-associated overexpressed, selectively in gastrointestinal tumors,especially in tumors of the colon. GPR35 is therefore suitable inparticular as molecular target structure for the diagnosis and treatmentof these tumors. Investigation to date of human GPR35, cf., for example,Horikawa Y, Oda N, Cox N J, Li X, Orho-Melander M, Hara M, Hinokio Y,Lindner T H, Mashima H, Schwarz P E, del Bosque-Plata L, Horikawa Y, OdaY, Yoshiuchi I, Colilla S, Polonsky K S, Wei S, Concannon P, Iwasaki N,Schulze J, Baier L J, Bogardus C, Groop L, Boerwinkle E, Hanis C L, BellG I Nat Genet. 2000 October; 26(2):163-75 suggested that GPR35 isactivated in many healthy tissues. By contrast, the investigationsaccording to the present technology showed that GPR35 is surprisinglynot significantly detectable in most normal tissues and, in contrastthereto, is highly activated in primary and metastatic colon tumors. Inaddition, besides the described GPR35 sequence, according to the presenttechnology a novel translation variant which makes use of an alternativestart codon has been found (SEQ ID NO: 10):

GPR35 is a member of the group of G-coupled receptors (GPCR), a verylarge protein family whose structure and function has been very wellinvestigated. GPCR are outstandingly suitable as target structures forthe development of pharmaceutically active substances, because themethods necessary therefor (e.g. receptor expression, purification,ligand screening, mutagenizing, functional inhibition, selection ofagonistic and antagonistic ligands, radiolabeling of ligands) is verywell developed and described in detail, cf., for example, “GProtein-Coupled Receptors” by Tatsuya Haga, Gabriel Berstein and GabrielBernstein ISBN: 0849333849 and in “Identification and Expression ofG-Protein Coupled Receptors Receptor Biochemistry and Methodology” byKevin R. Lynch ASIN: 0471183105. Realization according to the presenttechnology that GPR35 is undetectable in most healthy tissues butundergoes tumor-associated expression on the cell surface, enables it tobe used as tumor-associated target structure for example forpharmaceutically active ligands, especially in conjugation for examplewith radioactive molecules as pharmaceutical substances. It is possiblein a particular embodiment to use radiolabeled ligands which bind toGPR35 for detecting tumor cells or for treating colon tumors in vivo.

Example 2: Identification of GUCY2C in Hepatic and Ovarian Tumors andNovel GUCY2C Splice Variants as Diagnostic and Therapeutic CancerTargets

Guanylate cyclase 2C (SEQ ID NO: 2; translation product: SEQ ID NO:11)—a type I transmembrane protein—belongs to the family of natriureticpeptide receptors. The sequence is published in Genbank under theaccession number NM_004963. Binding of the peptides guanylin anduroguanylin or else heat-stable enterotoxins (STa) increases theintracellular cGMP concentration, thus inducing signal transductionprocesses inside the cell. Recent investigations indicate thatexpression of GUCY2C also extends to extraintestinal regions such as,for example, primary and metastatic adenocarcinomas of the stomach andof the esophagus (Park et al., Cancer Epidemiol Biomarkers Prev. 11:739-44, 2002). A splice variant of GUCYC which is found both in normaland transformed tissue of the intestine comprises a 142 bp deletion inexon 1, thus preventing translation of a GUCY2C-like product (Pearlmanet al., Dig. Dis. Sci. 45:298-05, 2000). The only splice variantdescribed to date leads to no translation product.

The aim according to the present technology was to identifytumor-associated splice variants for GUCY2C which can be utilized bothfor diagnosis and for therapy. RT-PCR investigations with aGUCY2C-specific primer pair (SEQ ID NO: 22, 23, 98, 99) show pronouncedexpression of GUCY2C transcripts in normal colon and stomach, and weakexpression in liver, testis, ovary, thymus, spleen, brain and lung (tab.2, FIG. 19). Expression in colon and stomach was at least 50 timeshigher than in all other normal tissues. Marked GUCY2C transcript levelswere detected in colon carcinoma and stomach carcinoma (tab. 2). Theseresults were specified by a quantitative PCR analysis and showedpronounced GUCY2C expression in normal colon, ileum, and in almost allcolon carcinoma samples investigated (FIG. 2, 19B). A massiveoverexpression was detectable in some colon carcinoma samples. Inaddition, expression is found in 7/10 stomach tumors. We alsosurprisingly found that the gene is activated in many other previouslyundescribed tumors, inter alia ovarian, breast, liver and prostatetumors (FIG. 19B, tab. 2).

TABLE 2 GUC2C expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain + Colon carcinoma +++ CerebellumPancreatic carcinoma − Myocardium Esophageal carcinoma − Skeletal muscle− Stomach carcinoma +++ Myocardium Bronchial carcinoma − Stomach +++Mammary carcinoma −+ Colon +++ Ovarian carcinoma + Pancreas −Endometrial carci Kidney − ENT tumors Liver + Renal cell carcinomaTestis ++ Prostate carcinoma + Thymus + Liver carcinoma + Breast −Ovary + Uterus + Skin Lung + Thyroid Lymph nodes − Spleen + PBMC −Prostate −

The following primer pairs were used to detect splice variants incolonic tissue and colon carcinoma tissue:

(SEQ ID NO: 24, 29) GUCY2C-118s/GUCY2C-498as; (SEQ ID NO: 25, 30)GUCY2C-621s/GUCY2C-1140as; (SEQ ID NO: 26, 31)GUCY2C-1450s/GUCY2C-1790as; (SEQ ID NO: 27, 32)GUCY2C-1993s/GUCY2C-2366as; (SEQ ID NO: 28, 33)GUCY2C-2717s/GUCY2C-3200as; (SEQ ID NO: 24, 30)GUCY2C-118s/GUCY2C-1140as; (SEQ ID NO: 25, 31)GUCY2C-621s/GUCY2C-1790as; (SEQ ID NO: 26, 32)GUCY2C-1450s/GUCY2C-2366as; (SEQ ID NO: 27, 33)GUCY2C-1993s/GUCY2C-3200as.

On investigation of splice variants in colon carcinoma tissue, threepreviously unknown forms were identified according to the presenttechnology.

-   -   a) A deletion of exon 3 (SEQ ID NO: 3) which leads to a variant        of GUCY2C which is only 111 amino acids long and in which the        asparagine at position 111 is replaced by a proline.    -   b) A deletion of exon 6 (SEQ ID NO: 4) which results in an        expression product 258 amino acids long. This would generate a        C-terminal neoepitope comprising 13 amino acids.    -   c) A variant in which the nucleotides at positions 1606-1614,        and the corresponding amino acids L(536), L(537) and Q(538), are        deleted (SEQ ID NO:5).

The splice variants according to the present technology with deletionsrespectively in exon 3 and exon 6 (SEQ ID NO: 3, 4) are distinguished inparticular by the translation products (SEQ ID NO: 12, 13) having notransmembrane domain. The result in the case of exon 6 deletion is aC-terminal neoepitope of 13 amino acids which shows no homologywhatsoever with previously known proteins. This neoepitope is thuspredestined to be a target structure for immunotherapy. The splicevariant of the present technology with base deletions at positions1606-1614 (SEQ ID NO: 5) and its translation product (SEQ ID NO: 14)likewise comprises a neoepitope. Antibodies for detecting GUCY2C proteinwere produced by immunizing rabbits. The following peptides were used topropagate these antibodies:

SEQ ID NO: 100:  HNGSYEISVLMMGNS (AA 31-45) SEQ ID NO: 101: NLPTPPTVENQQRLA (AA 1009-1023)Such antibodies can in principle be used for diagnostic and therapeuticpurposes.

In particular, the extracellular domain of GUCY2C (position of thepredicted extracellular domain from the sequence of SEQ ID NO: 11: AA454-1073 (SEQ ID NO: 102)) can be used according to the presenttechnology as target structure of monoclonal antibodies. However, thestructural prediction is somewhat ambiguous and not yet verifiedexperimentally, so that an alternative membrane orientation is alsoconceivable. In this case, amino acids 1-431 would be outside the celland be suitable as starting point for monoclonal antibodies. Theseantibodies bind specifically to the cell surface of tumor cells and canbe used both for diagnostic and for therapeutic methods. Overexpressionof GUCY2C, especially in the colon tumors, provides additional supportfor such a use. Sequences coding for proteins can moreover be usedaccording to the present technology as vaccine (RNA, DNA, peptides,protein) for inducing tumor-specific immune responses (T-cell- andB-cell-mediated immune responses).

It is moreover possible in accordance with the cellular function of theGUCY2C molecule to develop according to the present technologysubstances, especially small molecules, which modulate the function ofthe enzyme on tumor cells. The product of the enzymic reaction, cGMP, isa known cellular signal molecule with a wide variety of functions(Tremblay et al. Mol Cell Biochem 230, 31).

Example 3: Identification of SCGB3A2 as Diagnostic and TherapeuticCancer Target

SCGB3A2 (SEQ ID NO: 6) (translation product: SEQ ID NO: 15) belongs tothe secretoglobin gene family. The sequence is published in GenBankunder accession number NM_054023. SCGB3A2 (UGRP1) is a homodimericsecretory protein with a size of 17 kDa, which is expressed exclusivelyin the lung and in the spiracles (Niimi et al., Am J Hum Genet70:718-25, 2002). RT PCR investigations with a primer pair (SEQ ID NO:37, 38) confirmed selective expression in normal lung tissue. Lung- andtrachea-specific genes, e.g. for surfactant proteins, are highlydownregulated in malignant tumors during dedifferentiation and arenormally undetectable in lung tumors. It was surprisingly found thatSCGB3A2 is active in primary and metastatic lung tumors. Theinvestigations according to the present technology showed that SCGB3A2is strongly and frequently expressed in bronchial carcinomas (FIG. 4).All the other 23 normal tissues tested, apart from lung and trachea,show no expression (cf. FIG. 20).

This was additionally confirmed in a specific quantitative RT-PCR (SEQID NO: 103, 104) (FIG. 20) which additionally shows overexpression by atleast one log in more than 50% of bronchial carcinomas. The selectiveand high expression of SCGB3A2 in normal lung tissue and in lungcarcinoma biopsies can be used according to the present technology formolecular diagnostic methods such as RT-PCR for detecting disseminatingtumor cells in blood and bone marrow, sputum, bronchial aspirate orlavage and for detecting metastases in other tissues, e.g. in locallymph nodes. In the healthy lung, SCGB3A2 is secreted by specializedcells exclusively into the bronchi. Accordingly, it is not to beexpected that SCGB3A2 protein will be detectable in body fluids outsidethe respiratory tract in healthy individuals. By contrast, in particularmetastatic tumor cells secrete their protein products directly into thebloodstream. One aspect of the present technology therefore relates todetection of SCGB3A2 products in serum or plasma of patients via aspecific antibody assay as diagnostic finding for lung tumors.

Antibodies for detecting SCGB3A2 protein were produced by immunizingrabbits. The following peptides were used to propagate these antibodies:

SEQ ID NO: 105:  LINKVPLPVDKLAPL SEQ ID NO: 106:  SEAVKKLLEALSHLVAn SCGB3A2-specific reaction was detectable in immunofluorescence (FIG.21). As expected for a secreted protein, the distribution of SCGB3A2 inthe cell was assignable to the endoplasmic reticulum and secretiongranules (FIG. 21A). To check the specificity, the cells weretransfected in parallel with a plasmid that synthesizes an SCGB3A2-GFPfusion protein. Protein detection took place in this case via theautofluorescent GFP (green fluorescent protein) (FIG. 21B).Superimposition of the two fluorescence diagrams shows unambiguouslythat the immune serum specifically recognizes SCGB3A2 protein (FIG.21C). Such antibodies can be used according to the present technologyfor example in the form of immunoassays for diagnostic and therapeuticpurposes.

Example 4: Identification of Claudin-18A1 and Claudin-10 18A2 SpliceVariants as Diagnostic and Therapeutic Cancer Targets

The claudin-18 gene codes for a surface membrane molecule having 4transmembrane domains and intracellular N terminus and C terminus. Niimiand colleagues (Mol. Cell. Biol. 21:7380-90, 2001) describe two splicevariants of the murine and human claudin-18 which have been described asexpressed selectively in lung tissue. (claudin-18A1) and in stomachtissue (claudin-18A2), respectively. These variants differ in the Nterminus (FIG. 22).

It was investigated according to the present technology how far thesplice variants claudin-18A2 (SEQ ID NO: 7) and claudin-18A1 (SEQ ID NO:117), and their respective translation products (SEQ ID NO: 16 and 118),can be used as markers or therapeutic target structures for tumors. Aquantitative PCR able to distinguish between the two variants wasestablished by selecting A1-specific (SEQ ID NO: 109 110) andA2-specific (SEQ ID NO: 107 & 108) primer pairs. The A2 splice variantwas additionally tested with a second primer pair in a conventional PCR(SEQ ID NO: 39 & 40). The Al variant is described to be active only innormal lung. However, it was surprisingly found according to the presenttechnology that the Al variant is also active in the gastric mucosa.Stomach and lung are the only normal tissues showing significantactivation. All other normal tissues are negative for claudin-Al. Oninvestigating tumors, it was surprisingly found that claudin-Al ishighly activated in a large number of tumor tissues. Particularly strongexpression is to be found in stomach tumors, lung tumors, pancreaticcarcinomas, esophageal carcinomas (FIG. 23), ENT tumors and prostatecarcinomas. The claudin-Al expression levels in ENT, prostate,pancreatic and esophageal tumors are 100-10 000 higher than the levelsin the corresponding normal tissues. The oligonucleotides used toinvestigate the claudin-A2 splice variant specifically enable thistranscript to be amplified (SEQ ID NO: 39 & 40 and 107 & 108).Investigation revealed that the A2 splice variant is expressed in noneof the more than 20 normal tissues investigated apart from gastricmucosa and to a small extent also testis tissue. We have found that theA2 variant is also, like the Al variant, activated in many tumors(depicted by way of example in FIG. 24). These include stomach tumors(8/10), pancreatic tumors (6/6), esophageal carcinomas (5/10) and livercarcinomas. Although no activation of claudin-18A2 is detectable inhealthy lung, it was surprisingly found that some lung tumors expressthe A2.1 splice variant.

TABLE 3A Expression of claudin-18A2 in normal and tumor tissues. Normaltissues Expression Tumor type Expression Brain − Colon carcinoma −Cerebellum − Pancreatic carcinoma ++ Myocardium − Esophageal carcinoma++ Skeletal muscle − Gastric carcinoma +++ Endometrium − Bronchialcarcinoma ++ Stomach +++ Breast carcinoma − Colon − Ovarian carcinoma −Pancreas − Endometrial carcinoma n.i. Kidney − ENT tumors ++ Liver −Renal cell carcinoma − Testis + Prostate carcinoma − Thymus − Breast −Ovary − Uterus − Skin − Lung − Thyroid − Lymph nodes − Spleen − PBMC −Esophagus −

TABLE 3B Expression of claudin-18A1 in normal and tumor tissues. Normaltissues Expression Tumor type Expression Brain − Colon carcinoma −Cerebellum − Pancreatic carcinoma ++ Myocardium − Esophageal carcinoma++ Skeletal muscle − Gastric carcinoma +++ Endometrium − Bronchialcarcinoma ++ Stomach +++ Breast carcinoma + Colon − Ovarian carcinoman.i. Pancreas − Endometrial carcinoma n.i. Kidney − ENT tumors ++ Liver− Renal cell carcinoma − Testis + Prostate carcinoma ++ Thymus − Breast− Ovary − Uterus − Skin − Lung +++ Thyroid − Lymph nodes − Spleen − PBMC− Esophagus −

Conventional PCR as independent control investigation also confirmed theresults of the quantitative PCR. The oligonucleotides (SEQ ID NO: 39,40) used for this permit specific amplification of the A2 splicevariant. It was shown according to the present technology that 8/10gastric carcinomas and half of the tested pancreatic carcinomas showedstrong expression of this splice variant (FIG. 5). By contrast,expression is not detectable in other tissues by conventional PCR. Inparticular, there is no expression in lung, liver, blood, lymph nodes,breast tissue and kidney tissue (tab. 3).

The splice variants thus represent according to the present technologyhighly specific molecular markers for tumors of the uppergastrointestinal tract as well as lung tumors, ENT tumors, prostatecarcinomas and metastases thereof. These molecular markers can be usedaccording to the present technology for detecting tumor cells. Detectionof the tumors is possible according to the present technology with theoligonucleotides described (SEQ ID NO: 39, 40, 107-110). Particularlysuitable oligonucleotides are primer pairs of which at least one bindsunder stringent conditions to a segment of the transcript which is 180base pairs long and is specific for one (SEQ ID NO: 8) or the othersplice variant (SEQ ID NO: 119).

In order to confirm these data at the protein level, claudin-specificantibodies and immune sera were generated by immunizing animals. Theplasma membrane localization of claudin-18 and the protein topology wasconfirmed by analysis of the transmembrane domains with bioinformatictools (TMHMM, TMPRED) and immunofluorescence investigations of cellswhich expressed claudin-18 fusion proteins tagged with enhanced GFP.Claudin-18 has two extracellular domains. The N-terminal extracellulardomain differs in sequence in the two splice variants (SEQ ID NO: 111for Al and SEQ ID NO: 112 for A2). The C-terminal extracellular domainis identical for both variants (SEQ ID NO: 137). To date, no antibodieswhich bind to the extracellular domains of claudin-18 have yet beendescribed. According to the present technology, peptide epitopes whichare located extracellularly and are specific for variant A1 or A2 oroccur in both variants were selected for the immunization. Both variantsof claudin-18 have no conventional glycosylation motifs and theglycosylation of the protein was therefore not to be expected.Nevertheless, account was taken in the selection of the epitopes thatepitopes which comprise asparagine, serine, threonine are potentiallyglycosylated in rare cases even without conventional glycosylationsites. Glycosylation of an epitope may prevent the binding of anantibody specific for this epitope. Inter alia, epitopes were selectedaccording to the present technology so that the antibodies generatedthereby permit the glycosylation status of the antigen to bedistinguished. The following peptides, inter alia, were selected forproducing antibodies for the immunization: SEQ ID NO: 17: DQWSTQDLYN(N-terminal extracellular domain, A2-specific, binding independent ofglycosylation) SEQ ID NO: 18: NNPVTAVFNYQ (N-terminal extracellulardomain, A2-specific, binding mainly to unglycosylated form, N37) SEQ IDNO: 113: STQDLYNNPVTAVF (N-terminal extracellular domain, A2-specific,binding only to non-glycosylated form, N37) SEQ ID NO: 114: DMWSTQDLYDNP(N-terminal extracellular domain, A1-specific) SEQ ID NO: 115:CRPYFTILGLPA (N-terminal extracellular domain, mainly specific for Al)SEQ ID NO: 116: TNFWMSTANMYTG (C-terminal extracellular domain,recognizes both A1 and A2).

The data for the A2-specific antibody produced by immunization with SEQID NO: 17 are shown by way of example. The specific antibody can beutilized under various fixation conditions for immunofluorescenceinvestigations. With comparative stainings of RT-PCR-positive andnegative cell lines, in an amount which is readily detectable, thecorresponding protein can be specifically detected in the gastriccarcinoma cell lines typed as positive (FIG. 25). The endogenous proteinis membrane-located and forms relatively large focal aggregates on themembrane. This antibody was additionally employed for protein detectionin Western blotting. As expected, protein is detected only in stomachand in no other normal tissue, not even lung (FIG. 29). The comparativestaining of stomach tumors and adjacent normal stomach tissue frompatients surprisingly revealed that claudin-18 A2 has a smaller massweight in all stomach tumors in which this protein is detected (FIG. 30,left). It was found according to the present technology in a series ofexperiments that a band also appears at this level when lysate of normalstomach tissue is treated with the deglycosylating agent PNGase F (FIG.30, right). Whereas exclusively the glycosylated form of the A2 variantis detectable in all normal stomach tissues, A2 is detectable as such inmore than 60% of the investigated gastric carcinomas, in particularexclusively in the deglycosylated form. Although the A2 variant ofclaudin-18 is not detected in normal lung even at the protein level, itis to be found in bronchial carcinomas, as also previously in thequantitative RT-PCR. Once again, only the deglycosylated variant ispresent (FIG. 31). Antibodies which recognize the extracellular domainof the claudin-18-A2 splice variant have been produced according to thepresent technology. In addition, antibodies which selectively recognizethe N-terminal domain of the claudin-18-Al splice variant (FIG. 28) andantibodies which bind to both variants in the region of the C-terminalextracellular domain (FIG. 27) have been produced. It is possibleaccording to the present technology to use such antibodies fordiagnostic purposes, e.g. immunohistology (FIG. 32), but also fortherapeutic purposes as explained above. A further important aspectrelates to differentially glycosylated domains of claudin-18. Antibodieswhich exclusively bind to non-glycosylated epitopes have been producedaccording to the present technology. Claudin-18 itself is a highlyselective differentiating antigen for stomach tissue (A2) and for thelung and stomach (Al). Since it is evidently affected by changes in theglycosylation machinery in tumors, a particular, deglycosylated, variantof A2 is produced in tumors. This can be utilized diagnostically andtherapeutically. Immune sera such as the one described here (againstpeptide of SEQ ID NO: 17) can be utilized diagnostically for example inWestern blotting. Antibodies which are entirely unable to bind to theglycosylated epitope as obtained for example by immunization withpeptide of SEQ ID NO:113 (FIG. 26), can distinguish tumor tissue fromnormal tissue in the binding. It is possible in particular to employsuch antibodies therapeutically because they are highly selective. Theproduced antibodies can be used directly also for producing chimeric orhumanized recombinant antibodies. This can also take place directly withantibodies obtained from rabbits (concerning this, see J Biol Chem. 2000May 5; 275(18):13668-76 by Rader C, Ritter G, Nathan S, Elia M, Gout I,Jungbluth A A, Cohen L S, Welt S, Old L J, Barbas C F 3rd. “The rabbitantibody repertoire as a novel source for the generation of therapeutichuman antibodies”). For this purpose, lymphocytes from the immunizedanimals were preserved. The amino acids 1-47 (SEQ ID NO: 19 and 120)also represent particularly good epitopes for immunotherapeutic methodssuch as vaccines and the adoptive transfer of antigen-specific Tlymphocytes.

Example 5: Identification of SLC13A1 as Diagnostic and TherapeuticCancer Target

SLC13A1 belongs to the family of sodium sulfate cotransporters. Thehuman gene is, in contrast to the mouse homolog of this gene,selectively expressed in the kidney (Lee et al., Genomics 70:354-63).SLC13A1 codes for a protein of 595 amino acids and comprises 13 putativetransmembrane domains. Alternative splicing results in 4 differenttranscripts (SEQ ID NO: 41-44) and its corresponding translationproducts (SEQ ID NO: 45-48). It was investigated whether SLC13A1 can beused as marker for kidney tumors. Oligonucleotides (SEQ ID NO: 49, 50)which enable specific amplification of SLC13A1 were used for thispurpose.

TABLE 4 Expression of SLC13A1 in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colon carcinoma nd Cerebellumnd Pancreatic carcinoma nd Myocardium nd Esophageal carcinoma ndSkeletal muscle nd Gastric carcinoma nd Mycardium − Bronchial carcinomand Stomach − Breast carcinoma nd Colon − Ovarian carcinoma nd Pancreasnd Endometrial carcinoma nd Kidney +++ ENT tumors nd Liver − Renal cellcarcinoma +++ Testis + Prostate carcinoma nd Thymus − Breast − Ovary −Uterus nd Skin nd Lung − Thyroid − Lymph nodes − Spleen − PBMC − Sigmoid− Esophagus −

RT-PCR investigations with an SLC13A1-specific primer pair (SEQ ID NO:49, 50) confirmed virtually selective expression in the kidney, andshowed according to the present technology a high expression invirtually all (7/8) investigated renal cell carcinoma biopsies (tab. 4,FIG. 6). Quantitative RT-PCR with specific primers (SEQ ID NO: 121, 122)also confirmed these data (FIG. 34). Weak signals were detectable in thefollowing normal tissues: colon, stomach, testis, breast, liver andbrain. Expression in renal carcinomas was, however, at least 100 timeshigher than in all other normal tissues.

In order to analyse the subcellular localization of SLC13A1 in the cell,the protein was fused to eGFP as reporter molecule and, aftertransfection of the appropriate plasmid, expressed heterologously in 293cells. The localization was then analysed under the fluorescencemicroscope. Our data impressively confirmed that SLC13A1 is an integraltransmembrane molecule (FIG. 35).

Antibodies for detecting the SLC13A1 protein were produced by immunizingrabbits. The peptides of SEQ ID NO: 123 and 124 were used forpropagating these antibodies. Such antibodies can in principle be usedfor diagnostic and therapeutic purposes. The SLC13A1 protein has 13transmembrane domains and 7 extracellular regions. These extracellulardomains of SLC13A1 in particular can be used according to the presenttechnology as target structures for monoclonal antibodies. SLC13A1 isinvolved as channel protein in the transport of ions. The extracellulardomains of SLC13A1 in the healthy kidney are directed polarically in thedirection of the urinary tract (luminally). However, high molecularweight monoclonal antibodies employed therapeutically are not excretedinto the urinary tract, so that no binding to SLC13A1 takes place in thehealthy kidney. By contrast, the polarity of SLC13A1 is abolished intumor cells, and the protein is available for antibody targetingdirectly via the bloodstream. The pronounced expression and highincidence of SLC13A1 in renal cell carcinomas make this proteinaccording to the present technology a highly interesting diagnostic andtherapeutic marker. This includes according to the present technologythe detection of disseminated tumor cells in serum, bone marrow, urine,and detection of metastases in other organs by means of RT-PCR. It isadditionally possible to use the extracellular domains of SLC13A1according to the present technology as target structure forimmunodiagnosis and therapy by means of monoclonal antibodies. SLC13A1can moreover be employed according to the present technology as vaccine(RNA, DNA, protein, peptides) for inducing tumor-specific immuneresponses (T and B cell-mediated immune responses). This includesaccording to the present technology also the development of so-calledsmall compounds which modulate the biological activity of SLC13A1 andcan be employed for the therapy of renal tumors.

Example 6: Identification of CLCA1 as Diagnostic and Therapeutic CancerTarget

CLCA1 (SEQ ID NO: 51; translation product: SEQ ID NO: 60) belongs to thefamily of Ca⁺⁺-activated Cf channels. The sequence is published inGenbank under the accession No. NM 001285. CLCA1 is exclusivelyexpressed in the intestinal crypt epithelium and in the goblet cells(Gruber et al., Genomics 54:200-14, 1998). It was investigated whetherCLCA1 can be used as marker for colonic and gastric carcinoma.Oligonucleotides (SEQ ID NO: 67, 68) which enable specific amplificationof CLCA1 were used for this purpose. RT-ECR investigations with thisprimer set confirmed selective expression in the colon, and showedaccording to the present technology high expression in (3/7)investigated colonic and (1/3) investigated gastric carcinoma samples(FIG. 7). The other normal tissues showed no or only very weakexpression. This was additionally confirmed with a specific quantitativeRT-PCR (SEQ ID NO: 125, 126), in which case no expression could bedetected in the normal tissues analyzed (FIG. 36). Of the tumor samplesinvestigated in this experiment, 6/12 colonic carcinoma samples and 5/10gastric carcinoma samples were positive for CLCA1. Overall, expressionof the gene in tumors appears to be dysregulated. Besides samples withvery strong expression, CLCA1 was markedly downregulated in othersamples.

The protein is predicted to have 4 transmembrane domains with a total of2 extracellular regions. These extracellular domains of CLCA1 inparticular can be used according to the present technology as targetstructures for monoclonal antibodies.

The pronounced expression and high incidence of CLCA1 in gastric andcolonic carcinomas make this protein according to the present technologyan interesting diagnostic and therapeutic marker. This includesaccording to the present technology the detection of disseminated tumorcells in serum, bone marrow, urine, and detection of metastases in otherorgans by means of RT-PCR. It is additionally possible to use theextracellular domains of CLCA1 according to the present technology astarget structure for immunodiagnosis and therapy by means of monoclonalantibodies. CLCA1 can moreover be employed according to the presenttechnology as vaccine (RNA, DNA, protein, peptides) for inducingtumor-specific immune responses (T and B cell-mediated immuneresponses). This includes according to the present technology also thedevelopment of so-called small compounds which modulate the biologicalactivity as transport proteins of CLCA1 and can be employed for thetherapy of gastrointestinal tumors.

Example 7: Identification of FLJ21477 as Diagnostic and TherapeuticCancer Target

FLJ21477 (SEQ ID NO: 52) and its predicted translation product (SEQ IDNO: 61) was published as hypothetical protein in Genbank under theaccession No. NM_025153. It is an integral membrane protein havingATPase activity and 4 transmembrane domains, which is accordinglysuitable for therapy with specific antibodies. RT-PCR investigationswith FLJ21477-specific primers (SEQ ID NO: 69, 70) showed selectiveexpression in the colon, and additionally various levels of expressionin (7/12) investigated colonic carcinoma samples (FIG. 8). The othernormal tissues showed no expression. This was confirmed additionally bya specific quantitative RT-PCR (SEQ ID NO: 127, 128). FLJ21477-specificexpression was detectable both in colon (FIG. 37A) and in 11/12 ofcolonic carcinomas. Besides the expression in colon tissue, expressionwas additionally detectable in stomach tissue. In addition, under theconditions of the quantitative RT-PCR, the expression detectable inbrain, thymus and esophagus was distinctly weaker compared with colonand stomach (FIG. 37A). It was moreover additionally possible to detectFLJ21477-specific expression in the following tumor samples: stomach,pancreas, esophagus and liver. The protein is predicted to have 4transmembrane domains with a total of 2 extracellular regions. Theseextracellular domains of FLJ21477 in particular can be used according tothe present technology as' target structures for monoclonal antibodies.

The expression and the high incidence of FLJ21477 for gastric andcolonic carcinomas make this protein according to the present technologya valuable diagnostic and therapeutic marker. This includes according tothe present technology the detection of disseminated tumor cells inserum, bone marrow, urine, and the detection of metastases in otherorgans by means of RT-PCR. In addition, the extracellular domains ofFLJ21477 can be used according to the present technology as targetstructure for immunodiagnosis and therapy by means of monoclonalantibodies. In addition, FLJ21477 can be employed according to thepresent technology as vaccine (RNA, DNA, protein, peptides) for inducingtumor-specific immune responses (T and B cell-mediated immuneresponses).

Example 8: Identification of FLJ20694 as Diagnostic and TherapeuticCancer Target

FLJ20694 (SEQ ID NO: 53) and its translation product (SEQ ID NO: 62)were published as hypothetical protein in Genbank under accession No.NM_017928. This protein is an integral transmembrane molecule(transmembrane domain AA 33-54), very probably with thioredoxinfunction. RT-PCR investigations with FLJ20694-specific primers (SEQ IDNO: 71, 72) showed selective expression in the colon, and additionallyvarious levels of expression in (5/9) investigated colonic carcinomasamples (FIG. 9). The other normal tissues showed no expression. Thiswas additionally confirmed by a specific quantitative RT-PCR (SEQ ID NO:129, 130) (FIG. 38). FLJ29694 expression was undetectable in any othernormal tissue apart from colon and stomach (not analysed in the firstexperiment).

The protein is predicted to have one transmembrane domain with anextracellular region. These extracellular domains of FLJ20694 inparticular can be used according to the present technology as targetstructures for monoclonal antibodies.

In addition, FLJ20694 can be employed according to the presenttechnology as vaccine (RNA, DNA, protein, peptides) for inducingtumor-specific immune responses (T and B cell-mediated immuneresponses). This includes according to the present technology also thedevelopment of so-called small compounds which modulate the biologicalactivity of FLJ20694 and can be employed for the therapy ofgastrointestinal tumors.

Example 9: Identification of Von Ebner's Protein (c20orf114) asDiagnostic and Therapeutic Cancer Target

von Ebner's protein (SEQ ID NO:54) and its translation product (SEQ IDNO: 63) were published as Plunc-related protein of the upper airways andof the nasopharyngeal epithelium in Genbank under the accession No.AF364078. It was investigated according to the present technologywhether von Ebner's protein can be used as marker of lung carcinoma.Oligonucleotides (SEQ ID NO: 73, 74) which enable specific amplificationof Ebner's protein were used for this purpose. RT-PCR investigationswith this primer set showed selective expression in the lung and in(5/10) investigated lung carcinoma samples (FIG. 10). In the group ofnormal tissues there was also expression in the stomach. The othernormal tissues showed no expression.

Example 10: Identification of Plunc as Diagnostic and Therapeutic CancerTarget

Plunc (SEQ ID NO: 55) and its translation product (SEQ ID NO: 64) werepublished in Genbank under the accession No. NM 016583. Human Plunccodes for a protein of 256 amino acids and shows 72% homology with themurine Plunc protein (Single and Single, Biochem Biophys Acta1493:363-7, 2000). Expression of Plunc is confined to the trachea, theupper airways, nasopharyngeal epithelium and salivary gland.

It was investigated according to the present technology whether Plunccan be used as marker of lung carcinoma. Oligonucleotides (SEQ ID NO:75, 76) which enable specific amplification of Plunc were used for thispurpose.

RT-PCR investigations with this primer set showed selective expressionin the thymus, in the lung and in (6/10) investigated lung carcinomasamples (FIG. 11). Other normal tissues showed no expression.

Example 11: Identification of SLC26A9 as Diagnostic and TherapeuticCancer Target

SLC26A9 (SEQ ID NO: 56) and its translation product (SEQ ID NO: 65) werepublished in Genbank under the accession No. NM_134325. SLC26A9 belongsto the family of anion exchangers. Expression of SLC26A9 is confined tothe bronchiolar and alveolar epithelium of the lung (Lohi et al., J BiolChem 277:14246-54, 2002).

It was investigated whether SLC26A9 can be used as marker of lungcarcinoma. Oligonucleotides (SEQ ID NO: 77, 78) which enable specificamplification of SLC26A9 were used for this purpose. RT-PCRinvestigations with SLC26A9-specific primers (SEQ ID NO: 77, 78) showedselective expression in the lung and in all (13/13) investigated lungcarcinoma samples (FIG. 12). The other normal tissues showed noexpression, with the exception of the thyroid. It was possible inquantitative RT-PCR experiments with the primers of SEQ ID NO: 131 and132 firstly to confirm these results, and to obtain additionalinformation. It was possible in pooled samples of 4-5 tumor tissues todetect high expression levels for SLC26A9-specific RNA in lung, colon,pancreas and stomach tumors. SLC26A9 is member of a family oftransmembrane anion transporters. In the healthy lung, the protein isluminally directed in the direction of the airways and thus not directlyavailable to IgG antibodies from the blood. By contrast, the polarity ofthe protein is abolished in tumors. It is therefore possible accordingto the present technology to address SLC26A9 as therapeutic target usingmonoclonal antibodies in the defined tumors, inter alia lung tumors,gastric carcinomas, pancreatic carcinomas. The pronounced, highexpression and high incidence of SLC26A9 for lung, stomach, pancreaticand esophageal carcinomas make this protein according to the presenttechnology an excellent diagnostic and therapeutic marker. This includesaccording to the present technology the detection of disseminated tumorcells in serum, bone marrow and urine, and detection of metastases inother organs by means of RT-PCR. In addition, the extracellular domainsof SLC26A9 can be used according to the present technology as targetstructure for immunodiagnosis and therapy by means of monoclonalantibodies. It is additionally possible to employ SLC26A9 according tothe present technology as vaccine (RNA, DNA, protein, peptides) forinducing tumor-specific immune responses (T and B cell-mediated immuneresponses). This includes according to the present technology also thedevelopment of so-called small compounds which modulate the biologicalactivity of SLC26A9 and can be employed for the therapy of lung tumorsand gastrointestinal tumors.

Example 12: Identification of THC1005163 as Diagnostic and TherapeuticCancer Target

THC1005163 (SEQ ID NO: 57) is a gene fragment from the TIGR gene index.The gene is defined only in the 3′ region, while an ORF is lacking.RT-PCR investigations took place with a THC1005163-specific primer (SEQID NO: 79) and an oligo dT₁₈ primer which had a specific tag of 21specific bases at the 5′ end. This tag was examined using databasesearch programs for homology with known sequences. This specific primerwas initially employed in the cDNA synthesis in order to precludegenomic DNA contaminations. RT-PCR investigations with this primer setshowed expression in the stomach, ovary, lung and in (5/9) lungcarcinoma biopsies (FIG. 13). Other normal tissues showed no expression.

Example 13: Identification of LOC134288 as Diagnostic and TherapeuticCancer Target

LOC134288 (SEQ ID NO: 58) and its predicted translation product (SEQ IDNO: 66) were published in Genbank under accession No. XM_059703.

It was investigated according to the present technology whetherLOC134288 can be used as marker of renal cell carcinoma.Oligonucleotides (SEQ ID NO: 80, 81) which enable specific amplificationof LOC134288 were used for this purpose. RT-PCR investigations showedselective expression in the kidney and in (5/8) investigated renal cellcarcinoma biopsies (FIG. 14).

Example 14: Identification of THC943866 as Diagnostic and TherapeuticCancer Target

THC 943866 (SEQ ID NO: 59) is a gene fragment from the TIGR gene index.It was investigated whether THC943866 can be used as marker of renalcell carcinoma. Oligonucleotides (SEQ ID NO: 82, 83) which enablespecific amplification of THC943866 were used for this purpose.

RT-PCR investigations with THC943866-specific primers (SEQ ID NO: 82,83) showed selective expression in the kidney and in (4/8) investigatedrenal cell carcinoma biopsies (FIG. 15).

Example 15: Identification of FLJ21458 as Diagnostic and TherapeuticCancer Target

FLJ21458 (SEQ ID NO: 84) and its predicted translation product (SEQ IDNO: 85) were published in Genbank under the accession No. NM_034850.Sequence analyses revealed that the protein represents a new member ofthe butyrophillin family. Structural analyses revealed that itrepresents a type 1 transmembrane protein with an extracellularimmunoglobulin domain. Oligonucleotides (SEQ ID NO: 86, 87) which enablespecific amplification of FLJ21458 were used for investigatingexpression. RT-PCR investigations with FLJ21458-specific primers (SEQ IDNO: 86, 87) showed selective expression in colon and in (7/10)investigated colonic carcinoma biopsies (FIG. 16, tab. 5). QuantitativeRT-PCR with specific primers (SEQ ID NO: 133, 134) confirmed thisselective expression profile (FIG. 39). It was additionally possible inthe experiment to detect FLJ21458 gastrointestinal-specifically in thecolon, and in stomach, in the rectum and cecum and in testis. 7/11 colonmetastasis samples were also positive in the quantitative PCR.FLJ21458-specific expression was extended to other tumors, and aprotein-specific expression was detectable in stomach, pancreas andliver tumors (tab. 5). Antibodies for detecting FLJ21458 protein wereproduced by immunizing rabbits. The following peptides were used topropagate these antibodies:

SEQ ID NO: 135: QWQVFGPDKPVQAL

SEQ ID NO: 136: AKWKGPQGQDLSTDS

An FLJ21458-specific reaction was detectable in immuno-fluorescence(FIG. 40). To check the specificity of the antibodies, 293 cells weretransfected with a plasmid that codes for an FLJ21458-GFP fusionprotein. Specificity was demonstrated on the one hand by colocalizationinvestigations using the FLJ21458-specific antibody, and on the otherhand via the auto-fluorescent GFP. Superimposition of the twofluorescent diagrams showed unambiguously that the immune serumspecifically recognises FLJ21458 protein (FIG. 40a ). Owing to theoverexpression of the protein, the resultant cell staining was diffuseand did not allow unambiguous protein localization. For this reason, afurther immunofluorescence experiment was carried out with the stomachtumor-specific cell line Snu16 which expresses FLJ21458 endogenously(FIG. 4IB). The cells were stained with the FLJ21458-specific antiserumand with another antibody which recognizes the membrane proteinE-cadherin. The FLJ21458-specific antibody stains the cell membranes atleast weakly and is thus evidence that FLF21458 is localized in the cellmembrane.

Bioinformatic investigations showed that the protein encoded by FLJ21458represents a cell surface molecule and has an immunoglobulinsupermolecule domain. Selective expression of this surface moleculemakes it a good target for developing diagnostic methods for thedetection of tumor cells and therapeutic methods for the elimination oftumor cells.

The pronounced expression and high incidence of FLJ21458 for gastric andcolonic carcinomas make this protein according to the present technologya highly interesting diagnostic and therapeutic marker. This includesaccording to the present technology the detection of disseminated tumorcells in serum, bone marrow and urine, and the detection of metastasesin other organs by means of RT-PCR. It is additionally possible toemploy the extracellular domains of FLJ21458 according to the presenttechnology as target structure for immuno-diagnosis and therapy by meansof monoclonal antibodies. It is additionally possible to employ FLJ21458according to the present technology as vaccine (RNA, DNA, protein,peptides) for inducing tumor-specific immune responses (T and Bcell-mediated immune responses). This includes according to the presenttechnology also the development of so-called small compounds whichmodulate the biological activity of FLJ21458 and can be employed for thetherapy of gastrointestinal tumors.

TABLE 5 FLJ21458 expression in normal and tumor tissues Normal tissuesExpression Tumor type Expression Brain − Colonic carcinoma 7/10Cerebellum − Pancreatic carcinoma 5/6  Myocardium nd Esophagealcarcinoma nd Skeletel muscle − Gastric carcinoma 8/10 Mycardium −Bronchial carcinoma nd Stomach ++ Breast carcinoma nd Colon +++ Ovariancarcinoma nd Pancreas − Endometrial carcinoma nd Kidney − ENT tumors ndLiver − Renal cell carcinoma nd Testis ++ Prostate carcinoma nd Thymusnd Colonic metastases 7/11 Breast nd Liver carcinoma 5/8  Ovary − Uterus− Skin − Lung − Thyroid nd Lymph nodes − Spleen − PBMC − Adrenal ndEsophagus − Small intestine − Prostate −

The invention claimed is:
 1. A method of detecting a cancer cell,comprising: detecting the presence of or determining the quantity of atumor-associated antigen in a biological sample obtained from blood,bone marrow, or urine of a patient having or suspected of said cancer,and detecting the cancer cell based on the presence or quantity of thetumor-associated antigen in the biological sample, wherein thetumor-associated antigen is encoded by a nucleic acid sequence of SEQ IDNO:84; the detecting or determining comprises (i) contacting thebiological sample with a probe prepared by amplification with a primerset under conditions that allow the probe to hybridize to SEQ ID NO: 84,and (ii) detecting the formation of or determining the quantity of acomplex between the probe and SEQ ID NO: 84; wherein the primer setcomprises at least one sense primer and at least one antisense primer,wherein the at least one sense primer is selected from the groupconsisting of SEQ ID NOs: 86 and 133, and the at least one antisenseprimer is selected from the group consisting of SEQ ID NOs: 87 and 134;and wherein said cancer cell is from a cancer or tumor selected from thegroup consisting of colon cancer, rectal cancer, pancreatic cancer,stomach cancer, gastric cancer, liver cancer, and testicular cancer. 2.The method of claim 1, wherein detecting the cancer cell comprisesdetermining regression, course or onset of the cancer in the patient. 3.The method of claim 1, wherein the method comprises detection of thepresence of or determination of the quantity of the tumor-associatedantigen in a first sample at a first point in time and in a furthersample at a second point in time and a comparison of the presence of orquantity of the tumor-associated antigen in the first and secondsamples.
 4. The method of claim 1, wherein the probe is labeled with adetectable marker.
 5. The method of claim 1, wherein the sample is froma tissue biopsy.
 6. The method of claim 1, wherein the detecting ordetermining comprises real-time reverse-transcription polymerase chainreaction (RT-PCR).
 7. A method of detecting a cancer cell comprising:detecting the presence of or determining the quantity of atumor-associated antigen in a biological sample obtained from blood,bone marrow, or urine of a patient having or suspected of said cancer,and detecting the cancer cell based on the presence or quantity of thetumor-associated antigen in the biological sample, wherein thetumor-associated antigen is encoded by a nucleic acid sequence of SEQ IDNO:84; the detecting or determining comprises: (i) contacting thebiological sample with a primer set under conditions which amplifies SEQID NO: 84 from the biological sample, and (ii) detecting the amplifiednucleic acids, wherein the primer set comprises at least one senseprimer and at least one antisense primer, wherein the at least one senseprimer is selected from the group consisting of SEQ ID NOs: 86 and 133,and the at least one antisense primer is selected from the groupconsisting of SEQ ID NOs: 87 and 134; and wherein an increased amount ofamplified nucleic acids in the biological sample as compared to a normalsample without cancer indicates the patient has cancer.
 8. The method ofclaim 7, wherein detecting the cancer cell comprises determiningregression, course or onset of the cancer in the patient.
 9. The methodof claim 7, wherein the method comprises detection of the presence of ordetermination of the quantity of the tumor-associated antigen in a firstsample at a first point in time and in a further sample at a secondpoint in time and a comparison of the presence of or quantity of thetumor-associated antigen in the first and second samples.
 10. The methodof claim 7, wherein the sample is from a tissue biopsy.
 11. The methodof claim 7, wherein the detecting or determining comprises real-timereverse-transcription polymerase chain reaction (RT-PCR).